Bicyclic peptide

Abstract

The present invention relates to peptide ligands which are capable of binding natriuretic peptide receptor 3 (NPR3). In particular, the invention describes bicyclic peptide ligands which comprise a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold. The invention also includes pharmaceutical compositions and drug conjugates comprising said peptide ligands and the use of said peptide ligands in therapy.

Description

[0001] BICYCLIC PEPTIDE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to peptide ligands which are capable of binding natriuretic peptide receptor 3 (NPR3). In particular, the invention describes bicyclic peptide ligands which comprise a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold. The invention also includes pharmaceutical compositions and drug conjugates comprising said peptide ligands and the use of said peptide ligands in therapy.

[0004] BACKGROUND OF THE INVENTION

[0005] Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class forthe development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers etal. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 A2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin aVb3 (355 A2) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A2; Zhao etal. (2007), J Struct Biol 160 (1), 1-10).

[0006] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin.

[0007] Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzeneand related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0008] Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009 / 098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)e-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule scaffold.

[0009] The natriuretic peptide (NP) system is a key neurohormonal system, which acts to maintain cardiovascular homeostasis. It consists primarily of three genetically distinct, but structurally related peptides: atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). ANP is predominantly synthesized in the atria of the heart and released in response to atrial distension, whereas BNP is mainly produced and secreted in the ventricles following volume overload (Volpe et al. (2014) International Journal of Cardiology, 176(3): 630-639). CNP is mainly secreted by the vascular endothelium following stimulation by pro-inflammatory cytokines and endothelium-dependent agonists.

[0010] There are also three known receptors for natriuretic peptides. Natriuretic peptide receptor 1 (NPR1 ; also known as NPRA or guanylate cyclase A), NPR2 (also known as NPRB or guanylate cyclase B) and NPR3 (also known as NPRC or guanylate cyclase C). NPR1 binds both ANP and BNP, whereas NPR2 binds CNP. Binding of NPs to NPR1 and NPR2 activates membrane-bound particulate guanylate cyclase and leads to the stimulation of the intracellular cyclic guanosine monophosphate (cGMP)-dependent second messenger signalling cascade, which mediates the majority of the physiological actions of NPs, including natriuresis, diuresis, and vasodilation (Potter (2011) The FEBS Journal, 278:1808-1817). NPR3 binds and internalises all three NPs, which targets them for degradation by intracellular proteases.

[0011] Cardio-renal diseases such as hypertension, heart failure, and chronic kidney disease are highly prevalent and associated with significant morbidity and mortality. Enhancing the beneficial physiological effects of the NP system may represent an attractive therapeutic approach to counter the pathophysiological effects of these diseases. Therefore, there remains a need for modulators of the NP system.SUMMARY OF THE INVENTION

[0012] According to a first aspect of the invention, there is provided a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, said peptide ligand comprising a polypeptide comprising at least three reactive groups, wherein the polypeptide is attached to a molecular scaffold.

[0013] According to a further aspect of the invention, there is provided a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence as defined herein.

[0014] According to a further aspect of the invention, there is provided a pharmaceutical composition which comprises the peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as described herein, in combination with one or more pharmaceutically acceptable excipients.

[0015] According to a further aspect of the invention, there is provided a drug conjugate comprising a peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as described herein, conjugated to one or more effector and / or functional groups.

[0016] According to a further aspect of the invention, there is provided the peptide ligand, polypeptide, pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as described herein, for use in for use in therapy.

[0017] According to a further aspect of the invention, there is provided the peptide ligand, polypeptide, pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as described herein, for use in preventing, suppressing, or treating a disease or disorder mediated by the natriuretic peptide (NP) system.

[0018] According to a further aspect of the invention, there is provided the peptide ligand, polypeptide, pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as described herein, for use in preventing, suppressing, or treating renal disease.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 : Proof of concept study of NPR3 pentapeptide tool as a potential therapeutic strategy on top of standard of care.

[0021] Figure 2: Inhibition of NPR3 binding Bicycle molecules presented on phage binding to biotinylated NPR3 by BNP in an Alphascreen competition assay.

[0022] Figure 3: Inhibition of NPR3 binding Bicycle molecules presented on phage binding to biotinylated NPR3 by active site binder B8 in an Alphascreen competition assay.

[0023] Figure 4: Inhibition of NPR3 binding Bicycle molecules presented on phage binding to biotinylated NPR3 by active site binder B8 in an Alphascreen competition assay.DETAILED DESCRIPTION

[0024] Definitions

[0025] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et aL, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et aL, Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art.

[0026] The term “amino acid” in the context of the present disclosure is used in its broadest sense and is meant to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, along with a side chain (e.g., a R group) specific to each amino acid. In some embodiments, the amino acids refer to naturally occurring L a-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; l=lle; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, A. L., (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term “amino acid” further includes D-amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogues, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as p-amino acids. For example, analogues or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as do natural Phe or Pro, are included within the definition of amino acid. Such analogues and mimetics are referred to herein as "functional equivalents" of the respective amino acid. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983, which is incorporated herein by reference. The chemical properties of the 20 main amino acids are provided in Table A below.Table A - Chemical properties of amino acids

[0027]

[0028] The terms “polypeptide”, and “peptide” are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. Polypeptides can also undergo maturation or post-translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like.

[0029] Peptides and peptide ligands

[0030] The present invention provides polypeptides which are capable of binding to NPR3. A peptide capable of binding to NPR3 as provided herein may be comprised, for example, in a peptide ligand comprising said polypeptide covalently bound to a molecular scaffold (e.g. a molecular scaffold as described in more detail herein) such that two or more polypeptide loops are formed on the molecular scaffold. A peptide or peptide ligand may be attached directly or indirectly (e.g. via a linker) to one or more effector groups and / or functional groups, such as one or more cytotoxic agents, lipids, albumin binders, radiochelators or chromophores.

[0031] The invention provides a polypeptide of the general formula RG1-X1-RG2-X2-RG3, or a modified derivative thereof, or a pharmaceutically acceptable salt thereof; wherein RG1, RG2 and RG3 represent the three reactive groups as defined herein, Xi represents a first chain of natural or non-natural amino acids, and X2 represents a second chain of natural or non-natural amino acids. When the polypeptide is bound to a molecular scaffold at the three reactive groups to form a peptide ligand, Xi joins with said RG1 and RG2 groups to form the firstpolypeptide loop “Loop 1”, and X2 joins with said RG2 and RG3 groups to form the second polypeptide loop “Loop 2”.

[0032] Typically, Xi represents a chain of from 2 to 9 natural or non-natural amino acids. Typically, Xi represents a chain of from 2 to 8 natural or non-natural amino acids, more typically from 3 to 7 amino acids. Xi may represent a chain of 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, typically 2, 3, 5, 6, 7, or 8 amino acids, more typically 3, 5, 6 or 7 amino acids.

[0033] Typically, X2 represents a chain of from 2 to 9 natural or non-natural amino acids. Typically, X2 represents a chain of from 2 to 8 natural or non-natural amino acids. X2 may represent a chain of 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, typically 2, 3, 4, 5, 7, 8 or 9 amino acids, more typically 2, 4, 5, 7, or 8 amino acids.

[0034] Typically, the total number of amino acids present in the chains of amino acids represented by Xi and X2 is no more than 15, for example no more than 14, no more than 13, no more than 12 or no more than 11. Typically, the total number of amino acids present in the chains of amino acids represented by Xi and X2 is no more than 12, and more typically no more than 11. Typically, the total number of amino acids present in the chains of amino acids represented by Xi and X2 is at least 5, for example at least 6, at least 7, at least 8, or at least 9. Typically, the total number of amino acids present in the chains of amino acids represented by Xi and X2 is at least 9. In one embodiment, the total number of amino acids present in the chains of amino acids represented by Xi and X2 is from 5 to 15, typically from 7 to 15, more typically from 8 to 13, yet more typically from 9 to 12 and most typically from 9 to 11.

[0035] In one embodiment, Xi contains 2 amino acids, and X2 contains 7 amino acids.

[0036] In one embodiment, Xi contains 3 amino acids, and X2 contains 7, 8 or 9 amino acids, typically 7 or 8 amino acids. In one embodiment, Xi contains 3 amino acids, and X2 contains 7 amino acids. In one embodiment, Xi contains 3 amino acids, and X2 contains 8 amino acids. In one embodiment, Xi contains 3 amino acids, and X2 contains 9 amino acids.

[0037] In one embodiment, Xi contains 5 amino acids, and X2 contains 5, 6, or 7 amino acids, typically 5 or 6 amino acids, and more typically 5 amino acids. In one embodiment, Xi contains 5 amino acids, and X2 contains 5 amino acids. In one embodiment, Xi contains 5 amino acids, and X2 contains 6 amino acids. In one embodiment, Xi contains 5 amino acids, and X2 contains 7 amino acids.

[0038] In one embodiment, Xi contains 6 amino acids, and X2 contains 3, 4, 5 or 6 amino acids, typically 3, 4 or 5 amino acids, and more typically 4 or 5 amino acids. In one embodiment, Xi contains 6 amino acids, and X2 contains 3 amino acids. In one embodiment, Xi contains 6 amino acids, and X2 contains 4 amino acids. In one embodiment, Xi contains 6 amino acids, and X2 contains 5 amino acids. In one embodiment, Xi contains 6 amino acids, and X2 contains 6 amino acids.In one embodiment, Xi contains 7 amino acids, and X2 contains 2 amino acids.

[0039] In one embodiment, Xi contains 8 amino acids, and X2 contains 4 amino acids.

[0040] In one embodiment, X2 contains 2 amino acids, and Xi contains 7 amino acids.

[0041] In one embodiment, X2 contains 3 amino acids, and Xi contains 6 amino acids.

[0042] In one embodiment, X2 contains 4 amino acids, and Xi contains 6 or 8 amino acids, typically 6 amino acids. In one embodiment, X2 contains 4 amino acids, and Xi contains 6 amino acids. In one embodiment, X2 contains 4 amino acids, and Xi contains 8 amino acids.

[0043] In one embodiment, X2 contains 5 amino acids, and Xi contains 5 or 6 amino acids. In one embodiment, X2 contains 5 amino acids, and Xi contains 5 amino acids. In one embodiment, X2contains 5 amino acids, and Xi contains 6 amino acids.

[0044] In one embodiment, X2 contains 6 amino acids, and Xi contains 5 or 6 amino acids, typically 5 amino acids. In one embodiment, X2 contains 6 amino acids, and Xi contains 5 amino acids. In one embodiment, X2 contains 6 amino acids, and Xi contains 6 amino acids.

[0045] In one embodiment, X2 contains 7 amino acids, and Xi contains 2, 3 or 5 amino acids, typically 3 amino acids. In one embodiment, X2 contains 7 amino acids, and Xi contains 2 amino acids. In one embodiment, X2 contains 7 amino acids, and Xi contains 3 amino acids. In one embodiment, X2 contains 7 amino acids, and Xi contains 5 amino acids.

[0046] In one embodiment, X2 contains 8 amino acids, and Xi contains 3 amino acids.

[0047] In one embodiment, X2 contains 9 amino acids, and Xi contains 3 amino acids.

[0048] In some embodiments, Xi contains 2 amino acids. In some embodiments, when Xi contains 2 amino acids, the amino acids within Xi may be each independently selected from an amino acid comprising an aromatic group on their side chain, typically a heteroaromatic group. In some embodiments, when Xi contains 2 amino acids, Xi comprises a moiety of formula -AA1-AA2-. Typically, AA1 and AA2are the same. In some embodiments, AA1 is W. In some embodiments, AA2 is W.

[0049] In some embodiments, Xi contains 3 amino acids. In some embodiments when Xi contains 3 amino acids, the amino acids within Xi may be each independently selected from D, W, I, A, F, G, K, T, and L. Typically, the amino acids are each independently selected from A, F, G, K, T and L, more typically from A, F, G and T. In some embodiments, when Xi contains 3 amino acids, Xi comprises a moiety of formula -AA1-AA2-AA3. In some embodiments, AA1 is D, A, K or T, and is typically A, K or T, more typically A or T. In some embodiments, AA2 is W, F or T, and is typically F or T, more typically F. In some embodiments, AA3 is I, G or L, and is typically G or L, more typically G.

[0050] In some embodiments, Xi contains 4 amino acids. In some embodiments when Xi contains 4 amino acids, Xi is as defined above for when Xi contains 3 amino acids, butcontains one additional amino acid which may be inserted anywhere within the amino acid sequence of Xi.

[0051] In some embodiments, Xi contains 5 amino acids. In some embodiments when Xi contains 5 amino acids, the amino acids within Xi may be each independently selected from R, I, D, H, L, E, V, S, A and W. Typically, the amino acids are each independently selected from R, I, D, H, L, S, A and W, more typically from R, I, D, H and L. In some embodiments, when Xi contains 5 amino acids, Xi comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-. In some embodiments, AA1 is R or L, and is typically R. In some embodiments, AA2 is I, D or S, and is typically I or S, more typically I. In some embodiments, AA3 is D, E or A, and is typically D or A, more typically D. In some embodiments, AA4 is H, E or W, and is typically H or W, more typically H. In some embodiments, AA5 is L, V or H, and is typically L or H, more typically L.

[0052] In some embodiments, Xi contains 6 amino acids. In some embodiments when Xi contains 6 amino acids, the amino acids within Xi may be each independently selected from V, L, P, W, A, R, E, F, D, I, G, S, H, M, K, and N. Typically, the amino acids are each independently selected from P, L, D, R, I, G, S, H, M, K, N, and A, more typically from P, L, D, R, I, G, H, M, K, N, and A, and most typically from P, I, D, R, L, G, N and A. In some embodiments, when Xi contains 6 amino acids, Xi comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-. In some embodiments, AA1 is V, R, P, H or N, and is typically P, H or N, more typically P or N. In some embodiments, AA2 is L, E, I or R, and is typically L, I or R, more typically I. In some embodiments, AA3 is P, F D or M, and is typically D or M, more typically D. In some embodiments, AA4 is P, D, R or A, and is typically R, D or A, more typically R or A. In some embodiments, AA5 is W, L, I or K, and is typically L, I or K, more typically L or I. In some embodiments, AA5 comprises a non-polar, aliphatic side chain. In some embodiments, AAe is A, L, G, S or I, and is typically G, S or I, more typically G or I, and most typically is G.

[0053] In some embodiments, Xi contains 7 amino acids. In some embodiments when Xi contains 7 amino acids, the amino acids within Xi may be each independently selected from R, I, D, P, V, K, L, H, S, F and M. Typically the amino acids are each independently selected from R, I, D, P, V and K. In some embodiments, when Xi contains 7 amino acids, Xi comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-. In some embodiments, AA1 is an amino acid with a charged side chain, typically a positively charged side chain. Typically, AA1 is R. In some embodiments, AA2 is an amino acid with a non-polar, aliphatic side chain. Typically, AA2 is I or L, and is typically I. In some embodiments, AAsis an amino acid with a charged side chain, typically a negatively charged side chain. Typically, AA3 is D. In some embodiments, AA4 is P or H, and is typically P. In some embodiments, AA5 is an amino acidwith an uncharged side chain. Typically, AAs is I or S, more typically I. In some embodiments, AAe is an amino acid with a non-polar aliphatic and / or aromatic side chain. Typically, AAe is V or F, and is typically V. In some embodiments, AA? is K or M, and is typically K.

[0054] In some embodiments, Xi contains 8 amino acids. In some embodiments when Xi contains 8 amino acids, the amino acids with Xi may be each independently selected from E, L, W, T, H, N, S and F. In some embodiments, when Xi contains 8 amino acids, Xi comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-. In some embodiments, AA1 is an amino acid with a charged side chain, typically a negatively charged side chain. Typically, AA1 is E. In some embodiments, AA2 is an amino acid with a non-polar, aliphatic side chain. Typically, AA2is L. In some embodiments, AA3is an amino acid with an aromatic group in its side chain, typically a heteroaromatic group. Typically, AA3 is W. In some embodiments, AA4 is an amino acid with a polar, uncharged side chain. Typically, AA4 is T. In some embodiments, AA5 is an amino acid with a charged side chain, typically a positively charged side chain. Typically, AA5 is H. In some embodiments, AAe is an amino acid comprising an amide group in its side chain. Typically, AAe is N. In some embodiments, AA7 is an amino acid comprising a hydroxy group in its side chain. Typically, AA7 is S. In some embodiments, AAs is an amino acid comprising an aromatic group in its side chain. Typically, AAs is F.

[0055] In some embodiments, Xi contains 9 amino acids. In some embodiments when Xi contains 9 amino acids, Xi is as defined above for when Xi contains 8 amino acids, but contains one additional amino acid which may be inserted anywhere within the amino acid sequence of Xi.

[0056] In some embodiments, X2 contains 2 amino acids. In some embodiments when X2 contains 2 amino acids, the amino acids within X2 may be each independently selected from P, L, D and I. Typically, the amino acids are selected from P and L. In some embodiments, when X2 contains 2 amino acids, X2 comprises a moiety of formula -AA1-AA2-. In some embodiments, AA1 is P or D, and is typically P. In some embodiments, AA2 is an amino acid with a non-polar, aliphatic side chain. In some embodiments, AA2 is L or I, and is typically L.

[0057] In some embodiments, X2 contains 3 amino acids. In some embodiments when X2 contains 3 amino acids, the amino acids within X2 may be each independently selected from A, I, Y, G, R and L. Typically, the amino acids are selected from G, R and L. In some embodiments, when X2 contains 3 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-. In some embodiments, AA1 is an amino acid with an uncharged side chain. In some embodiments, AA1 is A or G, and is typically G. In some embodiments, AA2 is I or R, and is typically R. In some embodiments, AA3 is an amino acid with an uncharged side chain. In some embodiments, AA3 is Y or L, and is typically L.In some embodiments, X2 contains 4 amino acids. In some embodiments, when X2 contains 4 amino acids, the amino acids within X2 may be each independently selected from I, G, M, N, S, T, F, P and R. Typically, the amino acids are selected from S, T, F, N, P and R, and more typically from N, P, R and F. In some embodiments, when X2 contains 4 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-AA4-. In some embodiments, AA1 is an amino acid with an uncharged side chain. In some embodiments, AA1 is I, S or N, and is typically S or N, more typically N. In some embodiments, AA2 is an amino acid with an uncharged side chain. In some embodiments, AA2 is G, S or P, and is typically S or P, more typically P. In some embodiments, AA3 is M, T or R, and is typically T or R, more typically R. In some embodiments, AA4 is an amino acid with an uncharged side chain. In some embodiments, AA4 is N or F, and is typically F.

[0058] In some embodiments, X2 contains 5 amino acids. In some embodiments, when X2 contains 5 amino acids, the amino acids within X2 may be each independently selected from G, K, Y, T, L, S, N, F, M, and P. Typically, the amino acids are selected from G, K, Y, T, L, S, N and F. In some embodiments, when X2 contains 5 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-. In some embodiments, AA1 is an amino acid with an uncharged side chain. In some embodiments, AA1 is G, Y or M, and is typically G or Y. In some embodiments, AA2 is K, S or P, and is typically K or S. In some embodiments, AA3 is Y, K or S, and is typically Y or K. In some embodiments, AA4 is an amino acid is a polar, uncharged side chain. In some embodiments, AA4 is T, N or S, and is typically T or N. In some embodiments, AA5 is an amino acid with a non-polar, uncharged side chain. In some embodiments, AA5 is L or F.

[0059] In some embodiments, X2 contains 6 amino acids. In some embodiments, when X2 contains 6 amino acids, the amino acids within X2 may be each independently selected from K, P, D, W, F, S, L, R and I. Typically, the amino acids are selected from P, L, D, R, I and S. In some embodiments, when X2 contains 6 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-. In some embodiments, AA1 is K or P, and is typically P. In some embodiments, AA2 is an amino acid comprising an uncharged side chain. In some embodiments, AA2 is P or L, and is typically L. In some embodiments, AA3 is an amino acid with a charged side chain, typically a negatively charged side chain. In some embodiments, AA3 is D. In some embodiments, AA4 is W or R, and is typically R. In some embodiments, AA5 is an amino acid comprising an uncharged side chain. In some embodiments, AA5 is F or I, and is typically I. In some embodiments, AAe is an amino acid comprising a hydroxy group in its side chain. In some embodiments, AAe is S.In some embodiments, X2 contains 7 amino acids. In some embodiments, when X2 contains 7 amino acids, the amino acids within X2 may be each independently selected from Y, D, T, L, N, S, W, G, P, and I. Typically, the amino acids are selected from P, I, D and S. In some embodiments, when X2 contains 7 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-. In some embodiments, AA1 is an amino acid with an uncharged side chain. In some embodiments, AA1 is Y, L or P, and is typically P. In some embodiments, AA2 is D, S or I, and is typically I. In some embodiments, AA3 is T, Y or D, and is typically D. In some embodiments, AA4 is an amino acid with an uncharged side chain. In some embodiments, AA4 is T, L or P, and is typically P. In some embodiments, AA5 is an amino acid with a non-polar, aliphatic side chain. In some embodiments, AA5is L or I, and is typically I. In some embodiments, AAe is an amino acid with an uncharged side chain. In some embodiments, AAe is N, W or S, and is typically S. In some embodiments, AA7 is L, G or I, and is typically I.

[0060] In some embodiments, X2 contains 8 amino acids. In some embodiments when X2 contains 8 amino acids, the amino acids within X2 may be each independently selected from A, Y, L, T, K, G, P, D, R, S and H. Typically, the amino acids are selected from P, L, D, R, L, S, H and Y. In some embodiments, when X2 contains 8 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-. In some embodiments, AA1 is an amino acid with an uncharged side chain. In some embodiments, AA1 is A or P, and is typically P. In some embodiments, AA2 is an amino acid with an aliphatic and / or aromatic side chain. In some embodiments, AA2 is Y or L, and is typically L. In some embodiments, AA3 is L or D, and is typically D. In some embodiments, AA4 is A or R, and is typically R. In some embodiments, AA5 is an amino acid with an uncharged side chain. In some embodiments, AA5 is T or L, and is typically L. In some embodiments, AAe is K or S, and is typically S. In some embodiments, AA7 is G or H, and is typically H. In some embodiments, AAs is an amino acid with an aliphatic and / or aromatic side chain. In some embodiments, AAs is Y or L, and is typically Y.

[0061] In some embodiments, X2 contains 9 amino acids. In some embodiments when X2 contains 9 amino acids, the amino acids within X2 may be each independently selected from F, G, P, L, D, H and V. In some embodiments, when X2 contains 9 amino acids, X2 comprises a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-. In some embodiments, AA1 is an amino acid comprising an aromatic group in its side chain. In some embodiments, AA1 is F. In some embodiments, AA2 is G. In some embodiments, AA3 is G. In some embodiments, AA4 is P. In some embodiments, AA5 is an amino acid comprising a non-polar, aliphatic side chain. In some embodiments, AA5 is L. In some embodiments, AAe is an amino acid comprising a charged side chain, typically a negatively charged side chain. In someembodiments, AAe is D. In some embodiments, AA? is an amino acid comprising a charged side chain, typically a positively charged side chain. In some embodiments, AA? is H. In some embodiments, AAs is an amino acid comprising a non-polar, aliphatic side chain. In some embodiments, AAs is L. In some embodiments, AAg is an amino acid comprising an non-polar, aliphatic side chain. In some embodiments, AAg is V.

[0062] In one embodiment, Xi comprises or consists of an amino acid sequence selected from:

[0063] VLPPWA (SEQ ID NO: 19).

[0064] WW (SEQ ID NO: 20);

[0065] RIDHL (SEQ ID NO: 21);

[0066] LDEEV (SEQ ID NO: 22);

[0067] ELWTHNSF (SEQ ID NO: 23);

[0068] REFDLL (SEQ ID NO: 24);

[0069] DWI (SEQ ID NO: 25);

[0070] RIDPIVK (SEQ ID NO: 26);

[0071] PLDRIG (SEQ ID NO: 27);

[0072] AFG (SEQ ID NO: 28);

[0073] KTL (SEQ ID NO: 29);

[0074] RLDHSFM (SEQ ID NO: 30);

[0075] PIDRLG (SEQ ID NO: 31);

[0076] TFG (SEQ ID NO: 32);

[0077] LSAWH (SEQ ID NO: 33);

[0078] PLDRIS (SEQ ID NO: 34);

[0079] HRMDKI (SEQ ID NO: 35); and

[0080] NIDAIG (SEQ ID NO: 36);

[0081] or a variant thereof as described herein.

[0082] Typically, Xi comprises or consists of an amino acid sequence selected from:

[0083] RIDHL (SEQ ID NO: 21);

[0084] RIDPIVK (SEQ ID NO: 26);

[0085] PLDRIG (SEQ ID NO: 27);

[0086] AFG (SEQ ID NO: 28);

[0087] KTL (SEQ ID NO: 29);

[0088] RLDHSFM (SEQ ID NO: 30);

[0089] PIDRLG (SEQ ID NO: 31);

[0090] TFG (SEQ ID NO: 32);

[0091] LSAWH (SEQ ID NO: 33);PLDRIS (SEQ ID NO: 34);

[0092] HRMDKI (SEQ ID NO: 35); and

[0093] NIDAIG (SEQ ID NO: 36);

[0094] or a variant thereof as described herein.

[0095] More typically, Xi comprises or consists of an amino acid sequence selected from: RIDHL (SEQ ID NO: 21);

[0096] RIDPIVK (SEQ ID NO: 26);

[0097] PLDRIG (SEQ ID NO: 27);

[0098] AFG (SEQ ID NO: 28);

[0099] KTL (SEQ ID NO: 29);

[0100] RLDHSFM (SEQ ID NO: 30);

[0101] PIDRLG (SEQ ID NO: 31);

[0102] TFG (SEQ ID NO: 32);

[0103] HRMDKI (SEQ ID NO: 35); and

[0104] NIDAIG (SEQ ID NO: 36);

[0105] or a variant thereof as described herein.

[0106] Most typically, Xi comprises or consists of an amino acid sequence selected from: RIDHL (SEQ ID NO: 21);

[0107] RIDPIVK (SEQ ID NO: 26);

[0108] AFG (SEQ ID NO: 28);

[0109] PIDRLG (SEQ ID NO: 31);

[0110] TFG (SEQ ID NO: 32); and

[0111] NIDAIG (SEQ ID NO: 36);

[0112] or a variant thereof as described herein.

[0113] In one embodiment, X2 comprises or consists of an amino acid sequence selected from:

[0114] AIY (SEQ ID NO: 37);

[0115] YDTTLNL (SEQ ID NO: 38);

[0116] GKYTL (SEQ ID NO: 39);

[0117] LSYLLWG (SEQ ID NO: 40);

[0118] IGMN (SEQ ID NO: 41);

[0119] KPDWFS (SEQ ID NO: 42);

[0120] AYLATKGL (SEQ ID NO: 43);

[0121] PL (SEQ ID NO: 44);

[0122] GRL (SEQ ID NO: 45);

[0123] PLDRLSHY (SEQ ID NO: 46);FGGPLDHLV (SEQ ID NO: 47);

[0124] DI (SEQ ID NO: 48);

[0125] YSKNF (SEQ ID NO: 49);

[0126] PIDPISI (SEQ ID NO: 50);

[0127] PLDRIS (SEQ ID NO: 51);

[0128] SSTF (SEQ ID NO: 52);

[0129] MPSSF (SEQ ID NO: 53); and

[0130] NPRF (SEQ ID NO: 54);

[0131] or a variant thereof as described herein.

[0132] Typically, X2comprises or consists of an amino acid sequence selected from: GKYTL (SEQ ID NO: 39);

[0133] PL (SEQ ID NO: 44);

[0134] GRL (SEQ ID NO: 45);

[0135] PLDRLSHY (SEQ ID NO: 46);

[0136] FGGPLDHLV (SEQ ID NO: 47);

[0137] DI (SEQ ID NO: 48);

[0138] YSKNF (SEQ ID NO: 49);

[0139] PIDPISI (SEQ ID NO: 50);

[0140] PLDRIS (SEQ ID NO: 51);

[0141] SSTF (SEQ ID NO: 52);

[0142] MPSSF (SEQ ID NO: 53); and

[0143] NPRF (SEQ ID NO: 54)

[0144] or a variant thereof as described herein.

[0145] More typically, X2comprises or consists of an amino acid sequence selected from: GKYTL (SEQ ID NO: 39);

[0146] PL (SEQ ID NO: 44);

[0147] GRL (SEQ ID NO: 45);

[0148] PLDRLSHY (SEQ ID NO: 46);

[0149] FGGPLDHLV (SEQ ID NO: 47);

[0150] DI (SEQ ID NO: 48);

[0151] YSKNF (SEQ ID NO: 49);

[0152] PIDPISI (SEQ ID NO: 50);

[0153] MPSSF (SEQ ID NO: 53); and

[0154] NPRF (SEQ ID NO: 54);

[0155] or a variant thereof as described herein.

[0156] Most typically, X2comprises or consists of an amino acid sequence selected from:GKYTL (SEQ ID NO: 39);

[0157] PL (SEQ ID NO: 44);

[0158] PLDRLSHY (SEQ ID NO: 46);

[0159] YSKNF (SEQ ID NO: 49);

[0160] PIDPISI (SEQ ID NO: 50); and

[0161] NPRF (SEQ ID NO: 54);

[0162] or a variant thereof as described herein.

[0163] In a further embodiment, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 19-36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 37-54, or a variant thereof as described herein. In some embodiments, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 21 and 26-36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39 and 44-54, or a variant thereof as described herein. In some embodiments, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 21 , 26-32, 35 and 36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39, 44-50, 53 and 54, or a variant thereof as described herein. In some embodiments, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 21 , 26, 28, 31 , 32, and 36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39, 44, 46, 49, 50, and 54, or a variant thereof as described herein.

[0164] The reactive groups RG1, RG2 and RG3 provide attachment points to a molecular scaffold, and typically comprise functional groups found within the side chains of particular amino acids found in the peptide. Such reactive groups may be e.g. cysteine, homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), pCys ((R)-3-amino-3-mercaptopropanoic acid), penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid), N-alkyl-Dap (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid, or lysine, or any other suitable reactive group. The reactive groups RG1, RG2 and RG3 may be the same or different.

[0165] Typically, at least one, at least two, or all three of RG1, RG2 and RG3 comprise a cysteine residue. Typically, RG1, RG2 and RG3 each comprise cysteine residues.

[0166] In a further embodiment, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 19-36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 37-54, or a variant thereof as described herein, wherein RG1, RG2 and RG3 are cysteine residues. In some embodiments, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 21 and 26-36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39 and 44-54, or a variant thereof as described herein, wherein RG1, RG2 and RG3 are cysteine residues. In some embodiments, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 21, 26-32, 35 and 36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39, 44-50, 53 and 54, or a variant thereof as described herein, wherein RG1, RG2 and RG3 are cysteine residues. In some embodiments, Xi comprises or consists of an amino acid sequence selected from SEQ ID NOs: 21, 26, 28, 31, 32, and 36, or a variant thereof as described herein, and X2 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39, 44, 46, 49, 50, and 54, or a variant thereof as described herein, wherein RG1, RG2 and RG3 are cysteine residues.

[0167] In some embodiments, the polypeptide RG1-X1-RG2-X2-RG3 has an amino acid sequence selected from:

[0168] RG1-VLPPWA-RG2-CAIY-RG3 (SEQ ID NO: 55);

[0169] RG1-WW-RG2-YDTTLNL-RG3 (SEQ ID NO: 56);

[0170] RG1-RIDHL-RG2-GKYTL-RG3 (SEQ ID NO: 57);

[0171] RG1-LDEEV-RG2-LSYLLWG-RG3 (SEQ ID NO: 58);

[0172] RG1-ELWTHNSF-RG2-IGMN-RG3 (SEQ ID NO: 59);

[0173] RG1-REFDLL-RG2-KPDWFS-RG3 (SEQ ID NO: 60);

[0174] RG1-DWI-RG2-AYLATKGL-RG3 (SEQ ID NO: 61);

[0175] RG1-RIDPIVK-RG2-PL-RG3 (SEQ ID NO: 62);

[0176] RG1-PLDRIG-RG2-GRL-RG3 (SEQ ID NO: 63);

[0177] RG1-AFG-RG2-PLDRLSHY-RG3 (SEQ ID NO: 64);

[0178] RG1-KTL-RG2-FGGPLDHLV-RG3 (SEQ ID NO: 65);

[0179] RG1-RLDHSFM-RG2-DI-RG3 (SEQ ID NO: 66);

[0180] RG1-PIDRLG-RG2-YSKNF-RG3 (SEQ ID NO: 67);

[0181] RG1-TFG-RG2-PIDPISI-RG3 (SEQ ID NO: 68);

[0182] RG1-LSAWH-RG2-PLDRIS-RG3 (SEQ ID NO: 69);

[0183] RG1-PLDRIS-RG2-SSTF-RG3 (SEQ ID NO: 70);

[0184] RG1-HRMDKI-RG2-MPSSF-RG3 (SEQ ID NO: 71); and

[0185] RG1-NIDAIG-RG2-NPRF-RG3 (SEQ ID NO: 72),

[0186] or a variant thereof as described herein; wherein RG1, RG2 and RG3 are as defined herein.

[0187] In one embodiment, the polypeptide RGi-Xi-RG2-X2-RG3has an amino acid sequence selected from:

[0188] CVLPPWACAIYC (SEQ ID NO: 1);

[0189] CWWCYDTTLNLC (SEQ ID NO: 2);CRIDHLCGKYTLC (SEQ ID NO: 3);

[0190] CLDEEVCLSYLLWGC (SEQ ID NO: 4);

[0191] CELWTHNSFCIGMNC (SEQ ID NO: 5);

[0192] CREFDLLCKPDWFSC (SEQ ID NO: 6);

[0193] CDWICAYLATKGLC (SEQ ID NO: 7);

[0194] CRIDPIVKCPLC (SEQ ID NO: 8);

[0195] CPLDRIGCGRLC (SEQ ID NO: 9);

[0196] CAFGCPLDRLSHYC (SEQ ID NO: 10);

[0197] CKTLCFGGPLDHLVC (SEQ ID NO: 11);

[0198] CRLDHSFMCDIC (SEQ ID NO: 12);

[0199] CPIDRLGCYSKNFC (SEQ ID NO: 13);

[0200] CTFGCPIDPISIC (SEQ ID NO: 14);

[0201] CLSAWHCPLDRISC (SEQ ID NO: 15);

[0202] CPLDRISCSSTFC (SEQ ID NO: 16);

[0203] CHRMDKICMPSSFC (SEQ ID NO: 17); and

[0204] CNIDAIGCNPRFC (SEQ ID NO: 18),

[0205] or a variant thereof as described herein.

[0206] Typically, the polypeptide RG1-X1-RG2-X2-RG3 has an amino acid sequence selected from:

[0207] CRIDHLCGKYTLC (SEQ ID NO: 3);

[0208] CRIDPIVKCPLC (SEQ ID NO: 8);

[0209] CPLDRIGCGRLC (SEQ ID NO: 9);

[0210] CAFGCPLDRLSHYC (SEQ ID NO: 10);

[0211] CKTLCFGGPLDHLVC (SEQ ID NO: 11);

[0212] CRLDHSFMCDIC (SEQ ID NO: 12);

[0213] CPIDRLGCYSKNFC (SEQ ID NO: 13);

[0214] CTFGCPIDPISIC (SEQ ID NO: 14);

[0215] CLSAWHCPLDRISC (SEQ ID NO: 15);

[0216] CPLDRISCSSTFC (SEQ ID NO: 16);

[0217] CHRMDKICMPSSFC (SEQ ID NO: 17); and

[0218] CNIDAIGCNPRFC (SEQ ID NO: 18);

[0219] or a variant thereof as described herein.

[0220] More typically, the polypeptide RG1-X1-RG2-X2-RG3 has an amino acid sequence selected from:

[0221] CRIDHLCGKYTLC (SEQ ID NO: 3);

[0222] CRIDPIVKCPLC (SEQ ID NO: 8);CPLDRIGCGRLC (SEQ ID NO: 9);

[0223] CAFGCPLDRLSHYC (SEQ ID NO: 10);

[0224] CKTLCFGGPLDHLVC (SEQ ID NO: 11);

[0225] CRLDHSFMCDIC (SEQ ID NO: 12);

[0226] CPIDRLGCYSKNFC (SEQ ID NO: 13);

[0227] CTFGCPIDPISIC (SEQ ID NO: 14);

[0228] CHRMDKICMPSSFC (SEQ ID NO: 17); and

[0229] CNIDAIGCNPRFC (SEQ ID NO: 18);

[0230] or a variant thereof as described herein.

[0231] Most typically, the polypeptide RG1-X1-RG2-X2-RG3 has an amino acid sequence selected from:

[0232] CRIDHLCGKYTLC (SEQ ID NO: 3);

[0233] CRIDPIVKCPLC (SEQ ID NO: 8);

[0234] CAFGCPLDRLSHYC (SEQ ID NO: 10);

[0235] CPIDRLGCYSKNFC (SEQ ID NO: 13);

[0236] CTFGCPIDPISIC (SEQ ID NO: 14); and

[0237] CNIDAIGCNPRFC (SEQ ID NO: 18);

[0238] or a variant thereof as described herein.

[0239] The polypeptide RG1-X1-RG2-X2-RG3 may have an amino acid sequence according to any one of SEQ ID NO: 1 to 18, or 55 to 72, wherein one, two, three, four, or five of the amino acids, typically one, two or three of the amino acids, are modified as described herein. Alternatively, the polypeptide RG1-X1-RG2-X2-RG3 may comprise an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 1-18, or 55-72, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. It will be appreciated that references herein to “sequence identity” are to be understood as meaning the percentage identity between two protein sequences, e.g. SEQ ID NO: X and SEQ ID NO: Y, for example as measured using the methods described herein.

[0240] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0241] (a) B1.B-B-B-C-B-B-B2-B-B-B3.B-C,

[0242] wherein B is any amino acid,

[0243] Bi is C or Cam,

[0244] B2 is D or E,

[0245] B3 is A, D, S or Z-1 ; or(b) CTFGCPIDPISIC (SEQ ID NO: 14); or

[0246] (c) CTFGCPIDPISIC (SEQ ID NO: 14) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0247] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0248] (a) C-B1-B2-W-C-N-B3-B4-L-V-R-M-Y-C,

[0249] wherein Bi is A or E or S,

[0250] B2is A or P or S or T,

[0251] B3 is A L or M or P or S,

[0252] B4 is H or M or N; or

[0253] (b) CSSWCNSMLVRMYC; or

[0254] (c) CSSWCNSMLVRMYC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0255] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CDWICAYLATKGLC (SEQ ID NO: 7).

[0256] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0257] (a) C-B1-B2-B3-C-B4-L-D-B5-B6-B7-B8-B9-C,

[0258] wherein Bi is A or K,

[0259] B2is F or 2Nal,

[0260] B3 is G or K,

[0261] B4 is P or Hyp,

[0262] B5 is K or R,

[0263] Be is I or L,

[0264] B7 is S,

[0265] Bs is A or H,

[0266] Bg is V or Y;

[0267] (b) CAFGCPLDRLSHYC (SEQ ID NO: 10); or

[0268] (c) CAFGCPLDRLSHYC (SEQ ID NO: 10) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0269] (a) C-B-B-B1.B2.B-C-B-B-B-B-B-B3,

[0270] wherein B is any amino acid,

[0271] Bi is AspT or Cya or D or tetrazole-Ala,

[0272] B2 is Agb or H or NArg or Q,

[0273] B3 is C or NMe C;

[0274] (b) CRIDHLCGKYTLC (SEQ ID NO: 3); or

[0275] (c) CRIDHLCGKYTLC (SEQ ID NO: 3) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0276] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CKTMIRCPYPGKC.

[0277] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CGRPSSCYANKFC.

[0278] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CGGENRCPTKKWC.

[0279] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0280] (a) C-B1-L-P-B2-W-A-C-B3-I-Y-C,

[0281] wherein Bi is A or D or E or N or T or V,

[0282] B2 is D or I or L or P or Q or T or V,

[0283] B3 is A or D or E or Q or V;

[0284] (b) CVLPPWACAIYC (SEQ ID NO: 1); or

[0285] (c) CVLPPWACAIYC (SEQ ID NO: 1) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0286] (a) C-P-B1-D-R-I-G-C-B2-B3-B4-C,

[0287] wherein Bi is F or L or M,

[0288] B3 is G or R,

[0289] B4 is R or L or S or T or M or N,

[0290] B5 is A or F or L or M;

[0291] (b) CPLDRIGCGRLC (SEQ ID NO: 9); or

[0292] (c) CPLDRIGCGRLC (SEQ ID NO: 9) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0293] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPFDPIGCRHFC.

[0294] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0295] (a) C-N-B1-L-L-B2-K-C-D-W-F-B3-C,

[0296] wherein Bi is F or H or P or V or Y,

[0297] B2 is A or S,

[0298] B3 is N or Q or T;

[0299] (b) CNPLLAKCDWFQC; or

[0300] (c) CNPLLAKCDWFQC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0301] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0302] (a) C-P-B1-D-B2-L-G-C-N-B3-B4-M-C

[0303] wherein Bi is I or M,

[0304] B2 is R or P,

[0305] B3 is L or P,

[0306] B4 is R or G;(b) CPMDPLGCNLRMC; or

[0307] (c) CPMDPLGCNLRMC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0308] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPLDRISCSSTFC (SEQ ID NO: 16).

[0309] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CNIDAIGCNPRFC (SEQ ID NO: 18).

[0310] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0311] (a) B1.B-B-B2B-B3-B-B-C-B-B-B4,

[0312] wherein B is any amino acid,

[0313] Bi is C, meC, or [Im],

[0314] B2 aMeD or D or tetrazole,

[0315] B3 is Chg or EPA or F or I,

[0316] B4 is C or meC;

[0317] (b) CRIDPIVKCPLC (SEQ ID NO: 8); or

[0318] (c) CRIDPIVKCPLC (SEQ ID NO: 8) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0319] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CRLDHSFMCDIC (SEQ ID NO: 12).

[0320] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0321] (a) C-R-L-D-B1-B2-B3-B4-C-B5-L-C,

[0322] wherein Bi is P or Q,

[0323] B2 is I or L,

[0324] B3 is T or S,B4 is M or Q,

[0325] Bs is K or N;

[0326] (b) CRLDPLTQCKLC; or

[0327] (c) CRLDPLTQCKLC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0328] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CKVDHVRMCGLC.

[0329] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CSYEELKRVCLMPC.

[0330] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CDAWVCNYLRQKELC.

[0331] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0332] (a) C-B1-B2-W-B3-C-B4-Y-B5-B6-B7-B8-B9-B10-C,

[0333] wherein Bi is D or E,

[0334] B2 is D or E or N or P or Q,

[0335] B3 is I or V,

[0336] B4 is E or N or S or T,

[0337] B5 is I or L or M,

[0338] Be is H or N or R or S or T,

[0339] B7 is A or H or L or S,

[0340] Bs is K or M or R,

[0341] Bg is G or H or L or M or N,

[0342] B10 is L or M or N or S or V;

[0343] (b) CDDWVCTYMSARGVC; or

[0344] (c) CDDWVCTYMSARGVC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0345] (a) C-W-W-C-Y-D-B1-B2-B3-B4-B5-C,

[0346] wherein Bi or D or E or P or T,

[0347] B2 is A or E or S or T,

[0348] B3 is I or L or Q or V,

[0349] B4 is N or D,

[0350] B5is I or L or M;

[0351] (b) CWWCYDTTLNLC (SEQ ID NO: 2); or

[0352] (c) CWWCYDTTLNLC (SEQ ID NO: 2) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0353] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CLDEEVCLSYLLWGC (SEQ ID NO: 4).

[0354] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CELWTHNSFCIGMNC (SEQ ID NO: 5).

[0355] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CREFDLLCKPDWFSC (SEQ ID NO: 6).

[0356] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CKTLCFGGPLDHLVC (SEQ ID NO: 11).

[0357] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:

[0358] (a) C-B1-P-F-B2-C-R-I-D-B3-B4-B2-B5-B6-C,

[0359] wherein Bi is A or H or S

[0360] B2 is G or SB3 is K or R or T

[0361] B4 is I or L

[0362] B5 is A or L or S or T

[0363] Be is E or H or N or R;

[0364] (b) CHPFSCRIDKLSAEC; or

[0365] (c) CHPFSCRIDKLSAEC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

[0366] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPIDRLGCYSKNFC (SEQ ID NO: 13).

[0367] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPKMDRICIIEGFC.

[0368] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CLSAWHCPLDRISC (SEQ ID NO: 15).

[0369] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CHRMDKICMPSSFC (SEQ ID NO: 17).

[0370] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CRLDKLSCGRSSFC.

[0371] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises C-T-4FPhe-C-Hyp-tBuAla-D-P-tBuAla-S-l-C.

[0372] In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3) selected from the sequences listed in Tables 1 and 2. In one embodiment, provided herein is a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3) selected from the sequences listed in Tables 1 and 2 that does not comprise the N-terminus modification that is N-terminal to the 1stcysteine and / or that does not comprise theC-terminus modification that is C-terminal to the 3rdcysteine, or that comprises a different N-terminal or C-terminal modification. In certain embodiments, the ligand is not [lm]HLDPIRKCPLC-NH2. In certain embodiments, the ligand does not comprise cysteamine, or is not a ligand that comprises cysteamine.

[0373] In any of the above embodiments, alternative reactive groups may be used in place of the first, second and / or third cysteine residues. These reactive groups provide attachment points to a molecular scaffold, such as a molecular scaffold as defined herein; and typically comprise functional groups found within the side chains of particular amino acids found in the peptide. Suitable reactive groups may be e.g. cysteine, homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), pCys ((R)-3-amino-3-mercaptopropanoic acid), penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid), N-alkyl-Dap (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid, or lysine, or any other suitable reactive group. The reactive groups may be the same or different. In one embodiment, said reactive groups comprise cysteine residues. In another embodiment, one or more cysteines are substituted, for example with homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), PCys ((R)-3-amino-3-mercaptopropanoic acid), penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid), N-alkyl-Dap (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid, or lysine, or any other suitable reactive group. The reactive groups may be the same or different.

[0374] In some embodiments the polypeptide and / or peptide ligand is capable of binding to NPR3. In some embodiments the polypeptide and / or peptide ligand is specific to NPR3.

[0375] In one embodiment the polypeptide and / or peptide ligand competitively binds NPR3, such that it reduces or blocks the binding of a natural ligand (e.g., ANP, BNP and / or CNP) to NPR3. In one embodiment, the peptide ligand competitively binds NPR3, such that it reduces or blocks the binding of ANP to NPR3. In one embodiment, the peptide ligand competitively binds NPR3, such that it reduces or blocks the binding of BNP to NPR3. In one embodiment, the peptide ligand competitively binds NPR3, such that it reduces or blocks the binding of CNP to NPR3. In one embodiment, the peptide ligand competitively binds NPR3, such that it reduces or blocks the binding of ANP, BNP and CNP to NPR3. Competitive binding can be measured by methods known in the art, such as a competition assay as described in Examples 4-6.

[0376] In one embodiment, the peptide and / or peptide ligand is specific for (i.e. binds to) the active site of NPR3. Such an active site may in some embodiments comprise or define an epitope capable of being selectively bound by the provided peptides and ligands that comprise them. In one embodiment, the peptide and / or peptide ligand binds to the natriuretic peptides (NP) binding site of NPR3, for example the ANP, BNP or CNP binding site of NPR3. Theskilled person is able to identify the relevant binding site using routine techniques in the art, for example crystallography in the presence of a bound ligand, for example as described in He et al. 2006, J. Mol. Biol 361(4), 698-714). Without wishing to be bound by theory, it is thought that the bicyclic peptides ligands may bind to the ANP / BNP / CNP binding site of NPR3, i.e. to residues in the region of residue 65 to 190 of NPR3.

[0377] In some embodiments, the polypeptide comprising an amino acid sequence as described above, or a modified derivative thereof, or a salt thereof, is extended at the N-terminus. In some embodiments the polypeptide is extended at the N-terminus with one or more (e.g. from 1 to about 20, e.g. from 1 to about 10, or from 1 to about 5, e.g. 1, 2, 3, 4 or 5) additional amino acids or analogues thereof. In some embodiments the polypeptide is extended at the C-terminus. In some embodiments the polypeptide is extended at the C-terminus with one or more (e.g. from 1 to about 20, e.g. from 1 to about 10, or from 1 to about 5, e.g. 1 , 2, 3, 4 or 5) additional amino acids or analogues thereof. In some embodiments the polypeptide is extended at the N-terminus and the C-terminus. In some embodiments the polypeptide is extended at the N-terminus and the C-terminus with one or more (e.g. from 1 to about 20, or from 1 to about 10, e.g. from 1 to about 5, e.g. 1 , 2, 3, 4 or 5) additional amino acids or analogues thereof.

[0378] In some embodiments the polypeptide is modified at the N- and / or C-terminus. In some embodiments the polypeptide is modified at the N- and C- terminus. Examples of N-terminal modifications include extension of the peptide by one or more amino acids or amino acid analogues such as alanine (A) or a variant thereof including beta alanine (bA) or NMe-A, lysine (K) or a variant thereof including acetylated lysine “(Ac)K”, dK (“k”), K substituted with a diacid, or K substituted with ivDde or with PIB. The N-terminal modification may be N-terminal acetylation, represented by “Ac”, or amide formation with another carbonyl-containing alkyl derivative, typically a carboxylic acid. For example, “PhBu” N-terminal modification represents the addition of a phenylbutyric acid group to the terminal amine; “Ahx” N-terminal modification represents the addition of a 6-aminohexanoic acid group to the terminal amine etc. The N-terminal modification may include the addition of a spacer group (for example, 1 to 10 polyethylene glycol (PEG) groups e.g. 2 PEG groups) and / or the addition of a half-life extending portion (for example a C12-C18 diacid joined by a glutamic acid group). Typically, such N-terminal modifications are connected to the terminal amine by the formation of an amide bond with an acid group.

[0379] Examples of C-terminal modifications include extension of the peptide by one or more amino acids or amino acid analogues such as alanine (A) or variant thereof, lysine (K) or a variant thereof such as acetylated lysine “K(Ac)”, K(PIB), or C-terminal amidation, i.e. the conversion of a C-terminal carboxylic acid group (-CO(O)H or -CO(O)' to an amide -C(O)NH2.Amidation is typically indicated by -NH2 at the C-terminus of an amino acid sequence. The C-terminal modification may include the addition of a spacer group (for example, 1 to 10 polyethylene glycol (PEG) groups e.g. 2 PEG groups, or 1 to 10 sarcosine (Sar) groups). Typically, such C-terminal modifications are connected to the terminal acid by the formation of an amide bond with an amine group.

[0380] In one embodiment, the polypeptide additionally comprises an N-terminal addition which is an alanine residue, and a C-terminal addition which is an alanine residue. In one embodiment, the polypeptide additionally comprises an N-terminal acetyl group, and does not comprise a C-terminal addition. Typically, the C-terminus is amidated.

[0381] In one embodiment, the polypeptide additionally comprises N- and / or C-terminal additions and comprises an amino acid sequence which is selected from:

[0382] A-(SEQ ID NO: 1)-A;

[0383] A-(SEQ ID NO: 2)-A;

[0384] A-(SEQ ID NO: 3)-A;

[0385] A-(SEQ ID NO: 4)-A;

[0386] A-(SEQ ID NO: 5)-A;

[0387] A-(SEQ ID NO: 6)-A;

[0388] A-(SEQ ID NO: 7)-A;

[0389] A-(SEQ ID NO: 8)-A;

[0390] A-(SEQ ID NO: 9)-A;

[0391] Ac-(SEQ ID NO: 10);

[0392] A-(SEQ ID NO: 11)-A;

[0393] A-(SEQ ID NO: 12)-A;

[0394] A-(SEQ ID NO: 13)-A;

[0395] Ac-(SEQ ID NO: 14);

[0396] A-(SEQ ID NO: 15)-A;

[0397] A-(SEQ ID NO: 16)-A;

[0398] A-(SEQ ID NO: 17)-A; and

[0399] A-(SEQ ID NO: 18)-A;

[0400] wherein Ac- represents an N-terminal acetyl group, or a variant thereof as defined herein.

[0401] In some embodiments, a peptide ligand as provided herein comprises a polypeptide as provided herein attached to a molecular scaffold. The molecular scaffold can be any molecular scaffold as described in more detail herein. In some embodiments the molecular scaffold is attached to one or more reactive groups in the polypeptide. The reactive groups are typically functional groups found within the side chains of particular amino acids found inthe peptide. Such reactive groups may be a cysteine side chain, a homocysteine side chain, a p-cysteine side chain, a penicillamine side chain, a lysine side chain, or an N-terminal amine group (such as Dap or (N-alkyl)Dap e.g. (N-methyl)Dap) or any other suitable reactive group such as cysteamine or mercaptopropionic acid. In some embodiments the molecular scaffold is attached to one or more reactive groups e.g. to one or more cysteine, homocysteine, -cysteine, cysteamine, mercaptopropionic acid or penicillamine groups (e.g. to the thiol group comprised in the amino acid or amino acid analogue side chain) in the peptide. In some embodiments the molecular scaffold is attached to one or more reactive groups e.g. to one or more cysteine, groups (e.g. to the thiol group comprised in the cysteine side chain) in the peptide. In some embodiments the polypeptide comprises three cysteine residues and the molecular scaffold is attached to the three cysteine groups.

[0402] According to a further aspect of the invention, there is provided a bicyclic peptide ligand, or a pharmaceutically acceptable salt thereof, which comprises a peptide ligand as defined herein, and a molecular scaffold, wherein the three reactive groups (e.g. cysteine residues) of said peptide ligand form covalent bonds with the molecular scaffold to form two loop sequences.

[0403] According to a further aspect of the invention, there is provided a peptide ligand capable of binding NPR3, or a pharmaceutically acceptable salt thereof, said peptide ligand comprising a polypeptide comprising at least three reactive groups as defined herein, wherein the three reactive groups (e.g. cysteine residues) of said peptide ligand form covalent bonds with the molecular scaffold to form two loop sequences.

[0404] According to one embodiment of the invention, there is provided a peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, said peptide ligand having the structure of formula (I):

[0405]

[0406] wherein Scaffold represents the molecular scaffold as defined herein; and

[0407] Z1-RG1-X1-RG2-X2-RG3-Z2 represents the polypeptide, wherein RG1, RG2 and RG3 represent the three reactive groups as defined herein, Xi represents between 2 and 9 natural or nonnatural amino acids as defined herein which join with said RG1 and RG2 groups to form Loop1, X2 represents between 2 and 9 natural or non-natural amino acids as defined herein which join with said RG2 and RG3 groups to form Loop 2, such that the total number of amino acid residues within Xi and X2 does not exceed 12,

[0408] Z1 represents one or more optional N-terminal natural or non-natural amino acids or an acetyl group, and

[0409] Z2 represents one or more optional C-terminal natural or non-natural amino acids.

[0410] The polypeptide represented by RG1-X1-RG2-X2-RG3 may be as defined herein.

[0411] In one embodiment, Z1 represents an alanine residue or an acetyl group.

[0412] In one embodiment, Z2 is either absent or represents an alanine residue.

[0413] In a further embodiment, Xi comprises an amino acid sequence selected from SEQ ID NOs: 19-36, X2 comprises an amino acid sequence selected from SEQ ID NOs: 37-54, RG1, RG2 and RG3 comprise cysteine residues, Z1 represents an alanine residue or an acetyl group and Z2 is either absent or represents an alanine residue.

[0414] One example of a molecule suitable for use a molecular scaffold in the peptide ligands as provided herein is TATA (1,3,5-Triacryloylhexahydro-1,3,5-triazine, available from Sigma Aldrich). TATA has the structure:

[0415]

[0416] In some embodiments TATA reacts with thiol groups of amino acid side chains as described herein (for example cysteinyl-thiol groups of the peptides described herein) to form a peptide ligand comprising a scaffold which is a derivative of TATA of form:

[0417]

[0418] wherein each Cys-S represents a cysteine residue.

[0419] Accordingly, in one embodiment, the scaffold is a derivative of TATA which has the following structure:

[0420]

[0421] wherein * denotes the point of attachment of the three reactive groups RGi, RG2 and RG3.

[0422] In one embodiment, provided herein is a peptide ligand comprising a polypeptide as defined herein, or a modified derivative thereof, optionally extended at the N- and / or C-terminus, or a pharmaceutically acceptable salt thereof, bonded at each of three reactive groups (e.g. at each of the three cysteine residues) to a molecular scaffold which is a derivative of TATA as described herein.

[0423] In a further embodiment, the scaffold is a derivative of TATA which has the following structure:

[0424]

[0425] wherein * denotes the point of attachment of RG1, RG2 and RG3, and Z1-RG1-X1-RG2-X2-RG3- Z2 comprises an amino acid sequence which is selected from:

[0426] A-(SEQ ID NO: 1)-A (herein referred to as B1);

[0427] A-(SEQ ID NO: 2)-A (herein referred to as B2);

[0428] A-(SEQ ID NO: 3)-A (herein referred to as B3);

[0429] A-(SEQ ID NO: 4)-A (herein referred to as B4);

[0430] A-(SEQ ID NO: 5)-A (herein referred to as B5);

[0431] A-(SEQ ID NO: 6)-A (herein referred to as B6);

[0432] A-(SEQ ID NO: 7)-A (herein referred to as B7);

[0433] A-(SEQ ID NO: 8)-A (herein referred to as B8);

[0434] A-(SEQ ID NO: 9)-A (herein referred to as B9);

[0435] Ac-(SEQ ID NO: 10) (herein referred to as B10);

[0436] A-(SEQ ID NO: 11)-A (herein referred to as B11);

[0437] A-(SEQ ID NO: 12)-A (herein referred to as B12);A-(SEQ ID NO: 13)-A (herein referred to as B13);

[0438] Ac-(SEQ ID NO: 14) (herein referred to as B14);

[0439] A-(SEQ ID NO: 15)-A (herein referred to as B15);

[0440] A-(SEQ ID NO: 16)-A (herein referred to as B16);

[0441] A-(SEQ ID NO: 17)-A (herein referred to as B17); and

[0442] A-(SEQ ID NO: 18)-A (herein referred to as B18).

[0443] Numbering

[0444] When referring to amino acid residue positions within peptides of the invention, invariant residues are omitted from the numbering, therefore, the numbering of amino acid residues within peptides of the invention is referred to as below:

[0445] C-X1-X2-C-X3-X4-X5-X6-X7-X8-X9-C (SEQ ID NO: X).

[0446] Molecular Format

[0447] N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal pAla-Sar10-Ala tail would be denoted as:

[0448] PAIa-Sar10-A-(SEQ ID NO: X).

[0449] wherein Sar represents sarcosine, and pAla represents p-Alanine.

[0450] Inversed Peptide Sequences

[0451] In light of the disclosure in Nair et al (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus becomes C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).

[0452] Bicyclic Peptide Ligands

[0453] A bicyclic peptide ligand, as referred to herein, refers to a peptide ligand covalently bound to a molecular scaffold. Typically, such bicyclic peptides comprise a polypeptide having natural or non-natural amino acids, two or more reactive groups (e.g. cysteine, homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), pCys ((R)-3-amino-3-mercaptopropanoic acid), or penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid) or N-alkyl-Dap (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide is bound to the scaffold. In the present case, the peptides typically comprise at least three cysteine residues, and form at least two loops on the scaffold.

[0454] Accordingly, in some embodiments the present disclosure provides a peptide, a peptide ligand or a bicyclic peptide ligand as provided herein, wherein one or more of the cysteine residues of one or more of the polypeptides comprised in said peptide, peptide ligand, or bicyclic peptide ligand is replaced with homocysteine (hCys), pCys, penicillamine (Pen), Dap or N-methyl-Dap. In some embodiments one or more (e.g. 1, 2 or 3) of the cysteine residues in any one of the polypeptides described herein are replaced with homocysteine (hCys), PCys, penicillamine (Pen), Dap or N-methyl-Dap.

[0455] Peptide specificity

[0456] As explained above, in some embodiments the provided peptides and ligands comprising such (described in more detail herein) are specific for NPR3.

[0457] As used herein, the term “specific” (“specific binding”, etc) refers in its broadest sense to a peptide or peptide ligand which binds to its biological target. In some embodiments the peptide or peptide ligand binds to its biological target in a specific manner; that is, the binding to the biological target is not non-specific. In some embodiments a peptide which exhibits non-specific binding is promiscuous; that is the peptide is capable of binding to multiple different biological species, typically including the target of interest and off-target binding sites, such as binding sites on cell types other than a target cell type.

[0458] Accordingly, in some embodiments a peptide or peptide ligand which is chosen or designed to bind specifically to its intended target does not exhibit promiscuous binding to off-target binding sites.

[0459] In some embodiments the binding to the target is a binding to a particular epitope on the target. A peptide or peptide ligand may be designed to be specific for a particular epitope or may be identified by suitable screening methods, such as display techniques (e.g. phage display) which can be used to develop high-affinity binders against given targets (e.g. epitopes). Alternatively, a peptide or peptide ligand can be identified with specific binding to a biological target (such as a cellular target) without knowledge of the specific epitope to which it binds. In some embodiments a peptide or peptide ligand which specifically binds to a target or epitope has a high affinity for the target or epitope. In some embodiments the binding affinity of a peptide or peptide ligand to its epitope may be expressed in terms of itsdissociation constant (KD, also written as Kd). Typically, a peptide or peptide ligand which specifically binds to a biological target will have a KD against that target of less than 10 pM, e.g. less than 1 pM. Often, a peptide or peptide ligand which specifically binds to a biological target will have a nanomolar KD against that target, such as less than 100 nM, less than 20 nM, less than 10 nM, less than 5 nM or even less than 1 nM. Binding affinities can be determined by methods known in the art, such as SPR and competition assays. Some suitable assays are described in the examples.

[0460] In some embodiments a peptide or peptide ligand which specifically binds to a biological target will have a higher affinity (lower KD) for the specific biological binding site than for other biological binding site. For example, a peptide or peptide ligand which specifically binds to NPR3 will typically bind to the NP binding site of NPR3 with a higher affinity than to other binding sites. In some embodiments a peptide or peptide ligand which specifically binds to a biological target will bind to the desired binding site with an affinity at least twice as strong, e.g. at least 5 times, e.g. at least 10 times, e.g. at least 20 time, e.g. at least 50 times, e.g. at least 100 times, e.g. at least 1000 times or more as strong as the binding of the peptide to any other off-target binding sites.

[0461] Advantages of the Bicyclic Peptide Ligands

[0462] Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

[0463] Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamics and pharmacokinetic evaluation;

[0464] Protease stability. Bicyclic peptide ligands should ideally demonstrate stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a bicycle lead candidate can be developed in animal models as well as administered with confidence to humans;

[0465] Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra / inter-molecular H-bonding, which is important for formulation and absorption purposes;

[0466] An optimal plasma half-life in the circulation. Depending upon the clinical indication and treatment regimen, it may be required to develop a bicyclic peptide for short exposure in an acute illness management setting, or develop a bicyclic peptide with enhanced retention in the circulation, and is therefore optimal for the management of more chronic disease states. Other factors driving the desirable plasma half-life are requirements ofsustained exposure for maximal therapeutic efficiency versus the accompanying toxicology due to sustained exposure of the agent; and

[0467] Selectivity.

[0468] Pharmaceutically Acceptable Salts

[0469] It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.

[0470] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0471] Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

[0472] One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0473] If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO'), then a salt may be formed with an organic or inorganic base,generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+and K+, alkaline earth metal cations such as Ca2+and Mg2+, and other cations such as Al3+or Zn+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. , NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.

[0474] In one embodiment, the pharmaceutically acceptable salt is selected from the sodium, potassium, calcium or ammonium salt.

[0475] Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

[0476] The peptides (including peptide ligands comprising said peptides) of the invention may exist as a zwitterion. Such compounds may also be provided in the form of a pharmaceutically acceptable salt. Suitable salts include those formed with pharmaceutically acceptable acids, which provide a proton to a negatively charged group such as a COO' group, and a counterion to balance the positive charge on a positively charged group such as a quaternary nitrogen atom. Suitable pharmaceutically acceptable acids include hydrochloric acid, sulphonic acids including methanesulphonic acid and toluene sulphonic acid, ascorbic acid and citric acid. Hydrochloric acid and sulphonic acids are preferred, in particular hydrochloric acid. Alternatively, zwitterions can be combined with pharmaceutically acceptable bases, for example, alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides.

[0477] Modified Derivatives

[0478] It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention.

[0479] In some embodiments, modified derivatives include functional fragments, derivatives and variants of sequences provided herein.

[0480] As those skilled in the art will appreciate, fragments of amino acid sequences include deletion variants of such sequences wherein one or more, such as at least 1 , 2, 3, 4 or 5 aminoacids are deleted. Deletion may occur at the C- terminus or N-terminus of the reference sequence or within the reference sequence.

[0481] Derivatives of amino acid sequences include modified sequences including sequences which are modified in vivo or ex vivo. Many different protein modifications are known to those skilled in the art and include modifications to introduce new functionalities to amino acid residues, modifications to protect reactive amino acid residues or modifications to couple amino acid residues to chemical moieties such as reactive functional groups on linkers for attachment to such amino acid residues. Exemplary modifications that can be made to the provided peptides and ligands are described in more detail herein.

[0482] Derivatives of amino acid sequences include addition variants of such sequences wherein one or more, such as at least 1, 2, 3, 4, or 5 amino acids are added or introduced into the reference sequence. Addition may occur at the C- terminus or N-terminus of the reference sequence or within the reference sequence. Variants of amino acid sequences include sequences wherein one or more amino acid such as at least 1, 2, 3, 4, or 5 amino acid residues in the reference sequence are exchanged for one or more alternative residues. Variants of amino acid sequences include sequences carrying naturally occurring amino acids and / or unnatural amino acids.

[0483] Variants, derivatives and fragments of the aforementioned amino acid sequences typically retain at least some of the activity / functionality of the reference sequence. In a preferred embodiment, the variants, derivatives and fragments substantially retain their biological function(s) as described herein. Thus, in one embodiment the variants, derivatives and fragments retain the binding specificity of the reference sequence i.e. the ability to specifically bind to NPR3. In one such embodiment, the variants, derivatives and fragments bind to the same epitope as the reference sequence. In another embodiment, the variants, derivatives and fragments retain the binding affinity of the reference sequence. Typically, variants, derivatives and fragments of a reference sequence have increased / improved activity / functionality when compared to the reference sequence.

[0484] In some embodiments a variant, derivative or fragment of an amino acid sequence is expressed in terms of its percentage identity to the reference sequence. Protocols to determine percent identity are routine procedures within the scope of those skilled in the art. Suitable methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22(22) 4673-4680 (1994)) and iterative refinement (Gotoh, J. Mol. Biol. 264(4) 823-838 (1996)); and methods described by Altschul et aL, Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In an exemplary method, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff(ibid.), with percentage identity then calculated as: [100 x (T / L)]; wherein T = total number of identical matches and L = length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences.

[0485] In some embodiments a variant, derivative or fragment of a reference sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to the reference sequence.

[0486] Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues, or vice versa, replacement of one or more amino acids (e.g. one or more natural amino acids) with one or more isosteric and / or isolectronic amino acids, replacement of one or more natural amino acids with one or more isosteric and / or isoelectronic non-natural amino acids, or vice versa (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with an alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate / aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenolreactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.

[0487] Amino acid residues may typically be replaced with other amino acid residues of similar chemical structure, similar chemical properties or similar side-chain volume (“conservative substitutions”). The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table A above. Where amino acids have similarpolarity, this can also be determined by reference to the hydropathy scale for amino acid side chains, which is well-known the person skilled in the art.

[0488] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry, and / or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C-terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a lipid, an albumin binder, a radiochelator or a chromophore.

[0489] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.

[0490] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.

[0491] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

[0492] In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric / isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

[0493] Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca-disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

[0494] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine and / or the C-terminal cysteine.

[0495] In one embodiment, the modified derivative comprises replacement of one or moreoxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues.

[0496] In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

[0497] In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise 0-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

[0498] In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential proteolytic attack site(s).

[0499] In one embodiment, the modified derivative comprises replacement of one or more natural (i.e. canonical) amino acid residues with one or more non-natural (i.e. non-canonical) amino acid residues.

[0500] It should be noted that each of the above-mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

[0501] Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved;

[0502] Incorporating charged groups that exploit long-range ionic interactions, leading to faster on rates and to higher affinities (see for example Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and Incorporating additional constraint into the peptide, by for example constraining side chains of amino acids correctly such that loss in entropy is minimal upon target binding, constraining the torsional angles of the backbone such that loss in entropy is minimal upontarget binding and introducing additional cyclisations in the molecule for identical reasons. (for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor eta / , Curr. Medicinal Chem (2009), 16, 4399-418).

[0503] Isotopic Variations

[0504] The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

[0505] Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as2H (D) and3H (T), carbon, such as11C,13C and14C, chlorine, such as36CI, fluorine, such as18F, iodine, such as123l,125l and131l, nitrogen, such as13N and15N, oxygen, such as15O,17O and18O, phosphorus, such as32P, sulfur, such as35S, copper, such as64Cu, gallium, such as67Ga or68Ga, yttrium, such as90Y and lutetium, such as177Lu, and Bismuth, such as213Bi.

[0506] Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e.3H (T), and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0507] Substitution with heavier isotopes such as deuterium, i.e.2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0508] Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0509] Isotopically-labeled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processesanalogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0510] Molecular Scaffold

[0511] In some embodiments a polypeptide disclosed herein is attached to a molecular scaffold. Molecular scaffolds are described in, for example, WO 2009 / 098450 and references cited therein, particularly WO 2004 / 077062 and WO 2006 / 078161.

[0512] As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule. In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. References herein to “non-aromatic molecular scaffold” refers to any molecular scaffold as defined herein which does not contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system. Accordingly, in some embodiments the polypeptide is attached to a non-aromatic molecular scaffold. In other embodiments the polypeptide is attached to an aromatic molecular scaffold. Typically, the polypeptide is attached to a non-aromatic molecular scaffold.

[0513] In one embodiment the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0514] In one embodiment the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.

[0515] The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0516] The molecular scaffold of the invention contains chemical groups that allow functional groups of the polypeptide of the encoded library of the invention to form covalent links with the molecular scaffold. Said chemical groups are selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

[0517] Scaffold reactive groups that could be used on the molecular scaffold to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloalkanes).

[0518] Examples include bromomethylbenzene or iodoacetamide. Other scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides, a.p unsaturated carbonyl containing compounds and a-halomethylcarbonyl containing compounds. Examples of maleimides which may be used as molecular scaffolds in theinvention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene.

[0519] In a one embodiment, the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA)):

[0520]

[0521] Thus, following cyclisation with the bicyclic peptides of the invention (e.g. on the cysteine residues), the molecular scaffold forms a tri-substituted 1 ,1',1"-(1 ,3,5-triazinane-1,3,5-triyl)tripropan-1-one derivative of TATA having the following structure:

[0522]

[0523] wherein * denotes the point of attachment of the three reactive groups.

[0524] Reactive Groups

[0525] The molecular scaffold of the invention may be bonded to the polypeptide via functional or reactive groups on the polypeptide. These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. Such reactive groups may be a cysteine side chain, a homocysteine side chain (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), a pCys side chain ((R)-3-amino-3-mercaptopropanoic acid), a penicillamine side chain (Pen, (R)-2-amino-3-mercapto-3-methylbutanoic acid), a Dap group ((S)-2,3-diaminopropanoic acid), a N-alkyl-Dap group (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid, [a lysine side chain, or an N-terminal amine group or any other suitable reactive group. Detailsmay be found in WO 2009 / 098450. In one embodiment, the reactive groups are all cysteine residues.

[0526] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold / molecular core.

[0527] The polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.

[0528] In a preferred embodiment, polypeptides with three reactive groups are generated. Reaction of said polypeptides with a molecular scaffold / molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is favourable for several reasons. The nucleic acids of the compound libraries encode only the primary sequences of the polypeptide but not the isomeric state of the molecules that are formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer that was isolated in a screening or selection process. The formation of a single product isomer is also advantageous if a specific member of a library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.

[0529] In another embodiment of the invention, polypeptides with four reactive groups are generated. Reaction of said polypeptides with a molecular scaffold / molecular core having a tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomer can be determined by chemically synthesizing both isomers, separating the two isomers and testing both isomers for binding to a target ligand.

[0530] In one embodiment of the invention, at least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core. Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at leastthree bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved.

[0531] In another embodiment, the reactive groups of the polypeptide of the invention are reacted with molecular linkers wherein said linkers are capable to react with a molecular scaffold so that the linker will intervene between the molecular scaffold and the polypeptide in the final bonded state.

[0532] In some embodiments, amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid. Excluded from these exchangeable amino acids are the ones harbouring functional groups for cross-linking the polypeptides to a molecular core, such that the loop sequences alone are exchangeable. The exchangeable polypeptide sequences have either random sequences, constant sequences or sequences with random and constant amino acids. The amino acids with reactive groups are either located in defined positions within the polypeptide, since the position of these amino acids determines loop size.

[0533] In one embodiment, a polypeptide with three reactive groups has the sequence (X)iY(X)mY(X)nY(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 2 and 8 defining the length of intervening polypeptide segments, which may be the same or different, and I and o are numbers between 0 and 20 defining the length of flanking polypeptide segments.

[0534] Alternatives to thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively, these techniques may be used in modification or attachment of further moieties (such as small molecules of interest which are distinct from the molecular scaffold) to the polypeptide after they have been selected or isolated according to the present invention - in this embodiment then clearly the attachment need not be covalent and may embrace non-covalent attachment. These methods may be used instead of (or in combination with) the thiol mediated methods by producing phage that display proteins and peptides bearing unnatural amino acids with the requisite chemical reactive groups, in combination small molecules that bear the complementary reactive group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection / isolation phase. Further details can be found in WO 2009 / 098450 or Heinis, et aL, Nat Chem Biol 2009, 5 (7), 502-7.

[0535] Effector and Functional Groups

[0536] According to a further aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and / or functional groups.Effector and / or functional groups can be attached, for example, to the N and / or C termini of the polypeptide, to an amino acid within the polypeptide, or to the molecular scaffold.

[0537] Appropriate effector groups include antibodies and parts or fragments thereof. For instance, an effector group can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to the one or more constant region domains. An effector group may also comprise a hinge region of an antibody (such a region normally being found between the CH1 and CH2 domains of an IgG molecule).

[0538] Appropriate effector groups also include cytotoxic agents, lipids, albumin binders, radiochelators, and chromophores.

[0539] Functional groups include, in general, binding groups, drugs, reactive groups for the attachment of other entities, functional groups which aid uptake of the macrocyclic peptides into cells, and the like.

[0540] Synthesis

[0541] The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large-scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al. (supra).

[0542] Thus, the invention also relates to manufacture of polypeptides selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide made by chemical synthesis.

[0543] Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.

[0544] To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively, additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci U S A. 1994 Dec 20; 91 (26): 12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptide to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TATA orTATB) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptide, forming a disulfide -linked bicyclic peptide-peptide conjugate.

[0545] Similar techniques apply equally to the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

[0546] Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

[0547] In some embodiments a polypeptide as provided herein may be synthesised by solidphase synthesis. In some embodiments, the synthesis of a peptide ligand as provided herein may comprise solid-phase synthesis of a polypeptide as described herein. In some embodiments the solid-phase synthesis comprises Fmoc solid-phase peptide synthesis (e.g. as described in more detail herein). In some embodiments the synthesized polypeptide is cyclised with a molecular scaffold as described herein. In some embodiments the cyclised scaffold is purified e.g. by lyophilisation.

[0548] Pharmaceutical Compositions

[0549] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide or peptide ligand (for example a bicyclic peptide ligand) as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0550] Generally, the present peptides and peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

[0551] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives,such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0552] The compounds of the invention can be used alone or in combination with another agent or agents. The other agent for use in combination may be for example an angiotensinreceptor blocker (ARB) (e.g., irbesartan, valsartan, losartan and candesartan) or an angiotensin-converting enzyme (ACE) inhibitor (e.g. captopril, cilazapril, enalapril, ramipril, fosinopril, imidapril, lisinopril. Moexipril, perindopril, quinapril, ramipril, and trandolapril). The compounds of the invention can also be used in combination with biological therapies such as nucleic acid-based therapies, antibodies, bacteriophage or phage lysins.

[0553] The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. Preferably, the pharmaceutical compositions according to the invention will be administered parenterally. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

[0554] The peptides and peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.

[0555] The compositions containing the present peptides or peptide ligands or a cocktail thereof can be administered for therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generallyrange from 10 pg to 250 mg of selected peptide or peptide ligand per kilogram of body weight, with doses of between 100 pg to 25 mg / kg / dose being more commonly used.

[0556] A composition containing a peptide or peptide ligand according to the present invention may be utilised in therapeutic settings to treat a microbial infection or to provide prophylaxis to a subject at risk of infection e.g. undergoing surgery, chemotherapy, artificial ventilation or other condition or planned intervention. In addition, the peptides and peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

[0557] Therapeutic Uses

[0558] Natriuretic peptides (NPs) have been demonstrated to have beneficial cardiovascular and renal effects and have been investigated as therapeutic agents in a range of cardiovascular diseases including hypertension, heart failure, and acute myocardial infarction (Volpe et al. (2014) International Journal of Cardiology, 176(3): 630-639). Continuous intravenous administration of NPs has also been performed in cardiac surgery, resulting in cardiac and reno-protective effects.

[0559] NPR3 binds and internalises all three NPs, which targets them for degradation by intracellular proteases. Without being bound by theory, it is believed that the present NPR3-binding peptides and peptide ligands may be useful in therapy by blocking NPR3 and therefore selectively increasing NP signaling. Therefore, according to a further aspect of the invention, there is provided a peptide or peptide ligand (for example a bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein, for use in therapy. Also provided is a method of treating a patient in need thereof, which comprises administering to the patient the peptide, peptide ligand (for example bicyclic peptide ligand), or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein. Also provided is the use of a peptide, peptide ligand, (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, ordrug conjugate as defined herein, in the manufacture of a medicament.

[0560] According to a further aspect of the invention, there is provided the peptide, peptide ligand (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein, for use in preventing, suppressing, or treating a disease or disorder mediated by the natriuretic peptide (NP) system.According to a further aspect of the invention, there is provided a method of preventing, suppressing or treating a disease or disorder mediated by the natriuretic peptide (NP) system, which comprises administering to a patient in need thereof the peptide, peptide ligand (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein. According to a further aspect of the invention, there is provided the use of a peptide, peptide ligand, (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein, in the manufacture of a medicament for preventing, suppressing or treating a disease or disorder mediated by the natriuretic peptide (NP) system.

[0561] In one embodiment, the disease or disorder mediated by the natriuretic peptide (NP) system is a cardiovascular disease and / or a renal disease.

[0562] According to a further aspect of the invention, there is provided the peptide, peptide ligand (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein, for use in preventing, suppressing, or treating a cardiovascular disease and / or a renal disease, typically a renal disease. According to a further aspect of the invention, there is provided a method of preventing, suppressing or treating a cardiovascular disease and / or a renal disease, typically a renal disease, which comprises administering to a patient in need thereof the peptide, peptide ligand (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein. According to a further aspect of the invention, there is provided the use of a peptide, peptide ligand (for example bicyclic peptide ligand) or pharmaceutically acceptable salt thereof, pharmaceutical composition, or drug conjugate as defined herein, in the manufacture of a medicament for preventing, suppressing or treating a cardiovascular disease and / or a renal disease, typically a renal disease.

[0563] In one embodiment, the renal disease is chronic kidney disease (CKD).

[0564] Polypeptides and polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. Ligands having selected levels of specificity are useful in applications which involve testing in nonhuman animals, where cross-reactivity is desirable, or in diagnostic applications, where crossreactivity with homologues or paralogues needs to be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response to predetermined ranges of antigens can be exploited to tailor a vaccine to specific diseases and pathogens.Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

[0565] References herein to the term "prevention" involves administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become manifest.

[0566] Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available. The use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.

[0567] Abbreviations used throughout this application have the meaning set out below: a = [dA] or dAla

[0568] Abu = aminobutyric acid

[0569] ADMA = asymmetric dimethylarginine

[0570] Aib = 2-Aminoisobutyric acid

[0571] Agb or NArg = 2-Amino-4-guanidinobutanoic acid

[0572] agl = amino-gamma lactam

[0573] Agp = 2-amino-3-guanidinopropionic acid

[0574] Ahx = 6-Aminohexanoic acid

[0575] HO-C22-Ahx = 6-Aminohexanoic acid where the terminal amine is reacted with docosanedioic acid (C22 diacid)

[0576] aMeAsp = aMeD = a-methylaspartic acid

[0577] aMeF = a-methylphenylalanine

[0578] aMeLeu = a-methylleucine

[0579] Arg(Me) = arginine with terminal side chain amine methylated

[0580] AspT = tetrazole-alanine

[0581] Aze = Azetidine-2-carboxylic acid

[0582] -AE12AA-AE12AA-Lys(N3)-OH = C-terminus of peptide bonded to the following (number of PEG repeats may be altered, e.g. AE6AA represents PEGe):

[0583]

[0584] bAla or bA = beta alanine

[0585] Bip = (4-biphenyl)-alanine

[0586]

[0587] C5A = cyclopentylglycine

[0588] C18 = octadecanedioic acid

[0589] 4CyanoBu or 4-Cbn or Cbn = glycine substituted at CH2 position with -CH2CH2CN

[0590] Cam = cysteamine

[0591] Cba = cyclobutylalanine

[0592] CF3Ala = trifluoromethylalanine

[0593] Cha = 3-Cyclohexylalanine

[0594] Chg = cyclohexylglycine

[0595] CN3 = [CisAzP]; Cis-4-azido-L-proline

[0596] Cpa = cyclopropylalanine

[0597] cPentA or C5A = cyclopentylalanine

[0598] CpentG = cyclopentylglycine

[0599] CprG or Cpg = cyclopropylglycine

[0600] Cya = L-Cysteic acid

[0601] Cys(Acm) = cysteine where side chain thiol group is bonded to:

[0602]

[0603] [dA] or dAla = dAlanine

[0604] Dab = 2,4-Diaminobutanoic acid

[0605] Dap = Diaminopropionic acid

[0606] DBCO = Dibenzocyclooctyne[d R] or dArg = dArginine

[0607] EPA = 3-ethyl-pentanoic acid

[0608]

[0609] Haa = [HAA], 2-hydroxyacetic acid

[0610] HArg = homoarginine

[0611] hPhe or BnA = homophenylalanine, equivalent to benzylalanine

[0612] hGlu = homoglutamic acid

[0613] His(1Me) = 1 -methylhistidine

[0614] HLeu = homoleucine

[0615] HyP = hydroxyproline

[0616]

[0617] Ind = indole

[0618] ivDde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl

[0619] K(BOC) = tertbutyloxycarbonyl protected lysine

[0620] K(BPB) = lysine where the side chain amino group is reacted with (p-phenyl)phenyl butyric acid

[0621] -K-gE-K(PIB)VitC = lysine where the side chain amino group is bonded to:

[0622]

[0623] K(PCB) = lysine where the side chain amino group is reacted with p-chlorophenyl butyric acid K(PCF3B) = lysine where the side chain amino group is reacted with p-(trifluoromethyl)phenyl butyric acid

[0624] K(PFB) = lysine where the side chain amino group is reacted with p-fluorophenyl butyric acidK(4-iPr-Chx) - lysine where the side chain amino group is reacted with 4-isopropyl-cyclohexanecarboxylic acid

[0625] K(5Br-lndole-2) = lysine where the side chain amino group is bonded to:

[0626]

[0627] lysine where the side chain amino group is bonded to:

[0628]

[0629] K(C[n]-COOH) or K(C[n] diacid) or K(C[n]OH) or K(C[n]) or K(C[n] FA) = lysine where the side chain amino group is reacted with Cndiacid

[0630] K(C18 diacid) or K(C18OH) = lysine where the side chain amino group is reacted with octadecanedioic acid (C18 diacid)

[0631] K(C22-OH) or K(C22-COOH) = lysine where the side chain amino group is reacted with docosanedioic acid (C22 diacid)

[0632] K(GC18)-OH) = lysine where the side chain amino group is bonded to:

[0633]

[0634] K(gGlu-Lys(N3)-PIB) = lysine where the side chain amino group is bonded to:

[0635]

[0636] K((gGlu-K(PIB)-PIB))-OH = lysine where the side chain amino group is bonded to:

[0637]

[0638] K(PB) = lysine where the side chain amino group is reacted with phenylbutyric acid K(PBB) = lysine where the side chain amino group is reacted with p-bromophenyl butyric acid K(Pent) = lysine where the side chain amino group is reacted with 4-pentynoic acid K(Ph-O-Ad) = lysine where the side chain amino group is bonded to:

[0639]

[0640] K(PIB) = lysine where the side chain amino group is reacted with p-lodophenyl butyric acid K(PIB-gGlu(OH)-PEG2-PEG2) = lysine where the side chain amino group is bonded to:

[0641]

[0642] K(PEG2-PEG2-PIB) = lysine where the side chain amino group bonded to:

[0643]

[0644] K(PMeB) - lysine where the side chain amino group is reacted with p-methylphenyl butyric acid

[0645] K(N3) = lysine where the side chain amino group is replaced by N3

[0646] (PtBuB) - lysine where the side chain amino group is reacted with p-(tertbutyl)phenyl butyric acid

[0647] K(Triaz-AF488) = lysine where the side chain amino group is bonded to:

[0648]

[0649] K(Peg12Triaz-AF488) [NB number of PEG units can be changed e.g. PEG24] = lysine where

[0650] the side chain amino group is bonded to:

[0651]

[0652] -Lys(DBCO-NH2)-NH2: C-terminal modification where C-terminal acid is amidated with the following group:

[0653]

[0654] Lys(Me) - lysine where the side chain amino group is methylated

[0655] Lys(N3-Peg2) = lysine where the side chain amino group is bonded to:

[0656]

[0657] Lys(N3-(Peg2)2) = lysine where the side chain amino group is bonded to:

[0658]

[0659] Lys(C18_3insaturations) = lysine where the side chain amino group is bonded to:

[0660]

[0661] MerPro = mercaptopropionic acid

[0662] NArg = [Agb]; 2-Amino-4-guanidiniobutyric acid

[0663] Nle = norleucine

[0664] NMeAla = N-methylalanine

[0665] NMeArg or NMeR = N-methylarginine

[0666] NMeCys = meC = N-methylcysteine

[0667] NMeF = N-methylphenylalanine

[0668] NMelle = N-methylisoleucine

[0669] NMeLys = N-methyllysine

[0670] NMe-PEG2-PEG2 =

[0671]

[0672] NMe2bAla = N,N dimethyl beta-alanine

[0673] Orn = Ornithine

[0674] Palm = palmitic acid (C16 diacid)

[0675] PEG[n] = PEG polymer made of n ethylene glycol monomers

[0676] Phg = 2-Phenylglycine

[0677] PIB = 4-(p-lodophenyl)butyric acid

[0678]

[0679] Pro(PEG2)2-PIB = proline where the cyclic amino group is bonded to:

[0680]

[0681] Pro(PEG2)2-C18 =

[0682]

[0683] r = [dArg]

[0684] Sar or SAR = sarcosine

[0685] SDMA = Symmetric dimethylarginine

[0686] tBuAla or tBuA or Npg (neopentaglycine) = tertbutylalanine Tyr(Me) = O-methyl tyrosine

[0687] Z-1 = Lys(N3)-OH

[0688] Nal = naphthylalanine

[0689] 1Nal = 1-naphthylalanine

[0690] 2FPhe = 2-fluorophenylalanine

[0691] 2Nal = 2-naphthylalanine

[0692] 2Pal = 2-pyridinylalanine2,4FPhe = 2,4-difluorophenylalanine

[0693] 2Thi = 2-thienylalanine

[0694] 3FPhe = 3-fluorophenylalanine

[0695] 3Melle = 3-methylisoleucine (may also be referred to as EtMeMeA)

[0696] 3MePhe = 3-methylphenylalanine

[0697] 3,4FPhe = 3,4-difluorophenylalanine

[0698] 3,5FPhe = 3,5-difluorophenylalanine

[0699] 3Pal = 3-pyridinylalanine

[0700] 3Thi = 3-thienylalanine

[0701] 4Cbn = [CNbu]; 2-Amino-4-cyanobutanoid acid

[0702] 4CF3Phe = 4-(trifluoromethyl)phenylalanine

[0703] 4CIPhe = 4-chlorophenylalanine

[0704] 4CNPhe = 4-cyanophenylalanine

[0705] 4MePhe = 4-methylphenylalanine

[0706] 4OMePhe = 4-methoxyphenylalanine

[0707] 4FPhe = 4-fluorophenylalanine

[0708] 4GuanPhe or 4GuaPhe = 4-guanidinophenylalanine

[0709] 4-PhePro = 4-phenylproline

[0710] 4tBuPhe = 4-tertbutylphenylalanine

[0711] 4Pal = 4-pyridinylalanine

[0712] (4R)FPro = (4R)-fluoroproline

[0713] (4S)FPro = (4S)-fluoroproline

[0714] (4S)GuanPro = (4S)-guanidinoproline

[0715] (4R)GuanPro = (4R)-guanidinoproline

[0716] (4R)N3Pro or t(N3)P = (4R)-(N3)proline = 4-azido-L-proline

[0717] (4S)N3Pro or c(N3)P = (4S)-(N3)proline

[0718] (4R)NH2Pro = (4R)-aminoproline

[0719] (4S)NH2Pro = (4S)-aminoproline

[0720] (4,4)FPro = 4,4-difluoroproline

[0721] 55DMP = 5,5-dimethylproline

[0722] All publications, patents and patent applications cited herein are incorporated by reference in their entirety.

[0723] The invention is further described below with reference to the following examples.EXAMPLES

[0724] Example 1 : Proof of Concept Study of NPR3 blocking as potential therapeutic strategy A pentapeptide NPR3 receptor agonist with the below structure was prepared as set out in Veale et al. (2000) Bioorg. Med. Chem. Lett. 10:1949±1952.

[0725]

[0726] Male ZSF1 rats were used as a diabetic kidney injury model to assess potential therapeutic benefit with ACR as primary readout. Treatment groups included: lean (unchallenged lean rats) vehicle sc (subcutaneous), NPR3 (sc pentapeptide tool compound shown above), Losartan in dw (drinking water), vehicle in dw, and NPR3 + Losartan. Animals were placed in metabolic cages at baseline, after 4 and 9 weeks of treatment for urine collection and ACR assessment. As shown in Figures 1A and 1B, the NPR3 pentapeptide tool compound potentiates the beneficial effects of losartan on albuminuria (ACR) in ZSF1 rats and supports the hypothesis that NPR3 blocking is a potential therapeutic strategy on top of standard of care (SoC).

[0727]

[0728] General Method 1

[0729] Linear peptides were synthesized by solid-phase peptide synthesis (SPPS), based on Fmoc chemistry, either by manual coupling or using one of the following peptide synthesisers: SymphonyX, manufactured by Gyros Protein Technology or Syro II from Biotage and Biotage® I n itiator+ AlstraTM automated microwave peptide synthesizer. Rink Amide AM resin (100-200 mesh) or Wang Resin from IRIS Biotech with a loading between 0.4- 1.0 mmol / g was used unless otherwise stated. Standard Fmoc-amino acids were employed with the following side chain protecting groups: Arg(Pbf) or Arg(Boc)2; Asn(Trt); Asp(OtBu); Cys(Trt); Glu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu). The identity and purity of the products were determined by LCMS.

[0730] For linear peptide synthesis using a parallel synthesizer Syro II, the coupling reagent was HATU, diisopropylethylamine (DIPEA) was employed as a base, the capping step wasperformed using 5:6:89 v / v / v acetic anhydride / 2,6-lutidine / NMP and deprotection was achieved with 20 to 40% piperidine in DMF. Syntheses were performed using 0.51 mmol / g PS Fmoc-MBHA Rink amide AM resin, Fmoc-amino acids were utilised at a four-fold excess, coupling reagent at a 3.8-fold excess and base was at a two-fold excess with respect to the amino acids. Amino acids were dissolved at 0.5 M in DMF, HATU at 0.48 M in DMF, and DIPEA at 2 M in N-methylpyrrolidone (NMP). All amino acids were coupled twice, coupling times were 40 minutes at rt. Capping times were 5 min at rt and deprotection times 1 x 3 minutes with 40% piperidine in DMF followed by 1 x 10 minutes with 20% piperidine in DMF. A final capping step was performed when N-terminus acetylation or a specific capping group, linker handle or fluorophore labelling desired. After synthesis, the resin was washed with the following solvents: DCM, MeOH, DCM, methanol and Et20 before being dried under vacuum.

[0731] Cleavage of side-chain protecting groups and from the support was effected using 95:2.5:2.5:2.5 v / v / v / v TFA / H2O / iPr3SiH / 3,6-dioxa-1 ,8-octanedithiol (DODT) for 2-3 hours. Following cleavage, the spent resin was removed by filtration, and the filtrate was added to diethylether that had been cooled at 4 °C. Peptide pellet was centrifuged, the etheric supernatant discarded, and the peptide pellet washed with cold ether two more times. Peptides were then resolubilized in CHsCN-water 1:1 v / v acidified with 0.1% TFA and lyophilised. A small sample was removed for analysis of purity of the crude product by ultra- high performance liquid chromatography tandem mass spectrometry (UPLC-MS).

[0732] Following TFA-based cleavage from the resin, peptides were precipitated with diethyl ether and dissolved in 50:50 acetonitrile / water. The crude peptides (at ~1 mM concentration) were then cyclized with 1.3 equiv. of the scaffold, using ammonium bicarbonate (100 mM) as a base. Completion of cyclization was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) or LC-MS. Once complete, the cyclization reaction was quenched using N-acetyl cysteine (10 equiv. with respect to the peptide), and the solutions were lyophilized. The residue was dissolved in an appropriate solvent and purified by RP- HPLC. Peptide fractions of sufficient purity and the correct molecular weight (verified by either MALDI-TOF and HPLC or LC-MS) were pooled and lyophilized. Concentrations were determined by UV absorption using the extinction coefficient at 280 nm, which was based on Trp / Tyr content.

[0733] All amino acids, unless noted otherwise, were used in the L-configurations. References herein to amino acids with a “d” prefix (i.e. dC or dA) refer to amino acids in the D- configurations.

[0734]

[0735] >Method 2b. Synthesis of C-terminal modified bicycle peptides: in relation with ivDde manual— > Lipidation

[0736] The ivDDe protective group was removed by addition of a solution of 5% hydrazine in DMF. A freshly prepared soluion of 5% hydrazine in DMF (1.5 mL) was added to the protected peptide on resin (70 mg, 0.035 mmol). After 2 min the solution was drained from the resin, then the solution of 5% hydrazin in DMF (1.5 mL) was added to the resin and drained after 5 min (this step repeated for total 5x). The resin was then filtered and washed with DMF (5 x 1.5 mL) followed by DCM (3 x 1.5 mL) and dried under vacuum.

[0737] Capping / lipidation on Lysine side-chain:

[0738] A solutions of a capping group / lipid (4 eq, 0.25 M in DMF), DIC (4 eq, 0.24 M in DMF) and DIPEA (8 eq, 1 M in NMP) or at 75 °C for 10 min using DIC (4 eq, 0.2 M in DMF) and oxyma (4 eq, 0.2 M in DMF) in Alstra peptide synthesizer. The resin was then filtered and washed with DMF (5x), MeOH (5x), CH2Cl2(5x) and Et20 (5x).

[0739] Alternatively, a mixture of capping group / lipid activated with NHS ester (1 eq) and DIPEA (4 eq) in DMF were directly added to the resin and the reaction was agitated at room temperature for 1-2 h. The resin was then filtered and washed with DMF (5x), MeOH (5x), CH2CI2(5X) and Et2O (5x).

[0740]

[0741] Alexa Fluor 488 Azide (0.59 pmol, 0.8 eq.) in degassed DMF (0.060 mL) was added to the the peptide-alkyne (0.73 pmol, 1 eq). CuSO4 (0.5 mg, 2.2 mmol, 3 eq) in degassed water (0.060 mL) was added to ascorbic acid (1.3 mg, 7.3 mmol, 10 eq) and the resulting solution was added to the azide / peptide-alkyne mixture and the reaction mixture was stirred at room temperature (rt) protected from light for 40 min. The reaction mixture was then purified by revered phase HPLC to give the target compound (-46%).

[0742] Method 3a. Fmoc-Lys(Lipid)-OH buildinp block

[0743] / ) N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4-(4-iodophenyl)butanoyl)-L-lysine

[0744] DCC (1.104 g, 5.35 mmol) was added to a solution of 4-(4-iodophenyl)butanoic acid (1.45 g, 5.00 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (0.616 g, 5.35 mmol) in EtOAc (30 mL) and 1 ,4-dioxane (30 mL) at 0 °C. The reaction mixture was stirred at rt overnight and was then filtered (Celite) with the aid of EtOAc. The filtrate was concentrated in vacuo and the residue was partitioned between EtOAc and saturated NaHCO3(aq). The organic phase was washed with H2O and saturated NaCI(aq), dried (Na2SO4) and concentrated in vacuo. CH2CI2 was added to the residue and the solution was filtered (syringe filter) and concentrated in vacuo to give the crude desired compound 2,5-dioxopyrrolidin-1-yl 4-(4-iodophenyl)butanoate(1.965 g, quantitative) as a yellow solid. The material was taken to the next step without further purifications.

[0745] 1H NMR (500 MHz, CDCI3) 57.50 - 7.69 (m, 2H), 6.96 (d, 2H), 2.84 (s, 4H), 2.68 (t, 2H), 2.60 (t, 2H), 1.99 - 2.09 (m, 2H).

[0746] A solution of crude 2,5-dioxopyrrolidin-1-yl 4-(4-iodophenyl)butanoate (1.936 g, 5.00 mmol) in CH2CI2 (30 mL) was slowly (15 min) added to a suspension of (((9H-fluoren-9- yl)methoxy)carbonyl)-L-lysine (1.861 g, 5.05 mmol) and DIPEA (2.61 ml, 15.0 mmol) in CH2CI2 (30 mL). The reaction mixture was stirred at rt overnight and was then concentrated in vacuo. The residue was partitioned between EtOAc and 10% citric acid(aq). The organic phase was washed with H2O and saturated NaCI(aq), dried (Na2SO4) and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc:HOAc 98:2) to give the title compound (2.430 g, 76 %) as a white foam.

[0747] 1H NMR (500 MHz, CD3OD) 57.80 (d, 2H), 7.67 (t, 2H), 7.56 (d, 2H), 7.39 (t, 2H), 7.27 - 7.34 (m, 2H), 6.95 (d, 2H), 4.27 -4.38 (m, 2H), 4.20 (t, 1H), 4.13 (dd, 1H), 3.17 (t, 2H), 2.55 (t, 2H), 2.16 (t, 2H), 1.81 - 1.94 (m, 3H), 1.65 - 1.77 (m, 1H), 1.28 - 1.60 (m, 4H).

[0748] MS m / z 639 [M - H]-

[0749] (ii) N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-( 18-(tert-butoxy)- 18-oxooctadecanoyl)-L-

[0750] The title compound was prepared from 18-(tert-butoxy)-18-oxooctadecanoic acid using essentially the same method as described for (i) above (2.87 g, 80%).

[0751] 1H NMR (500 MHz, MeOD: CDCI3 1:1) 0 7.75 (d, 2H), 7.62 (t, 2H), 7.37 (t, 2H), 7.29 (t, 2H), 4.31 - 4.39 (m, 2H), 4.16 - 4.23 (m, 2H), 3.15 (t, 2H), 2.18 (t, 2H), 2.12 (t, 2H), 1.80 - 1.91 (m, 1H), 1.65 - 1.76 (m, 1H), 1.47 - 1.62 (m, 6H), 1.42 (s, 9H), 1.18 - 1.32 (m, 26H).

[0752] MS 721 [M + H]+

[0753] <

[0754]

[0755] The title compound was prepared from 19-(tert-butoxy)-19-oxononadecanoic acid using essentially the same method as described for (i) above (2.74 g, 75%).

[0756] 1H NMR (500 MHz, CD3OD-CDCI3 1:1) 0 7.75 (d, 2H), 7.63 (t, 2H), 7.37 (t, 2H), 7.29 (t, 2H), 4.30 - 4.39 (m, 2H), 4.15 - 4.23 (m, 2H), 3.15 (t, 2H), 2.18 (t, 2H), 2.12 (t, 2H), 1.80 - 1.90 (m, 1 H), 1.64 - 1.75 (m, 1 H), 1.47 - 1.61 (m, 6H), 1.42 (s, 9H), 1.21 - 1.29 (m, 28H).

[0757] MS 733 [M - H]-

[0758] Method 3b. C22- NHS ester22-((2, 5-dioxopyrrolidin- 1 -yl)oxy)-22-oxodocosanoic acid

[0759] Docosanedioic acid (0.5 g, 1.35 mmol), 1 -hydroxypyrrolidine-2, 5-dione (0.155 g, 1.35 mmol) and DMAP (0.8 mg, 7 pmol) in THF (17 mL) were stirred at rt for 10 min. DCC (0.278 g, 1.35 mmol) dissolved in THF (6 mL) was added dropwise during 30 min and the reaction mixture was then stirred for 24 h at rt. After filtration the mixture was evaporated. MeOH (5 mL) was added to the residue, heated to 45°C and was then stirred at rt for 1 h. The product was precipitated and was separated by filtration, washed with a small amount of MeOH and dried under reduced pressure to give the target compound (0.353 g, 56%).

[0760] 1H NMR (500 MHz, CDCI3) 52.83 (d, 4H), 2.59 (t, 2H), 2.34 (td, 2H), 1.70-1.79 (m, 2H), 1.58-1.68 (m, 2H), 1.19-1.44 (m, 32H).

[0761] Method 3c. Synthesis of Fmoc-NMePEG2-OH:

[0762] In a round bottom flask, 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (2.00 g, 5.19 mmol) was dissolved in CHCh (30 mL) and TFA (30 mL). The mixture was cooled down to 0 °C and then formaldehyde (4.68 ml, 62.3 mmol) was added. The mixture was then stirred at this temperature for 30 min then was allowed to warm up to rt and was stirred for 15 min. Then EtsSiH (16.58 ml, 103.8 mmol) was added and the reaction mixture was stirred for 15 min at rt. The reaction was cooled down to 0 °C for 15 min then it was stirred at rt for 30 min. The volatiles were evaporated and the residue was taken up with EtOAc. The organic solution was then washed with 10% NaHCO3(aq). The aqueous layer was then acidified with cone. HCI and extracted twice with EtOAc. The organics were combined and washed with saturated NaCI(aq). The organic layer was dried over (Na2SO4) and concentrated to afford the colorless oil 1-(9H-fluoren-9-yl)-4-methyl-3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (1.695 g, 82 %).

[0763] Method 4: Specific synthesis for (S)-1,5-dimethylimidazolidin-4-one-2-13C -CHLDPIRKCPLC-NH2 (Tata cyclized)

[0764] The tata cyclized peptide NMeAla-CHLDPIRKCPLC-NH2 (4.6 mg, 2.2 mmol), prepared using the general procedures, was dissolved in H2O (0.49 mL) and an aqueous solution of NH4HCO3 (10 mL, 2 M) was added. Formaldehyde-13C solution (20% in water) was diluted with water (1:1000) and this solution was added in small portions (in total: 0.53 mL) to the reaction mixture during 3 h and the reaction was monitored by LCMS. Upon completion the reaction mixture was freeze dried and used without further purification.

[0765] HRMS: Calcd for C7513CHi26N23Oi8S33+581.9610 [M + 3H]3+, found 581.9642.Example 3: SPR Binding Assay

[0766] Peptide affinities to biotinylated ectodomain of human natriuretic peptide receptor C / 3 (NPR3(48-485)-His) was determined in a direct binding assay using 8K or S200 surface plasmon resonance (SPR) biosensor (Cytiva, Sweden) at 20°C. Briefly, biotinylated NPR3 was immobilized on a biotin-coated sensor chip (Xantec, Germany) via SwitchAvidin (BioMediTech, Finland). The surface was washed with 10mM NaOH, 1M NaCI prior to immobilization of protein. NPR3 was preincubated with SwitchAvidin in a 2:1 ratio for 5 min before being diluted to 100 nM and immobilized. Immobilization levels were typically 5000 RU. The reference spot was treated as described, omitting the injection of protein. Peptide concentration series was injected over the immobilized protein in increasing concentrations using multi cycle (MCK) or single cycle kinetics (SCK) in running buffer (10 mM HEPES, 150 mM NaCI, 0.05% Tween20, 0.1% DMSO, pH 7.4). A 1:1 Langmuir interaction model was fitted to the experimental traces, enabling determination of Kon, KOff and Kd. The same procedure was also used for mouse and rat NPR3 to understand cross species affinity as well as NPR1 and NPR2 to understand receptor selectivity.

[0767] Table 1: Affinity KD Values Determined by SPR

[0768]

[0769]

[0770] Table 2: Affinity KD Values Determined by SPR

[0771] Note - in the table below, any N-terminal “H-“ should be disregarded and ignored.

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798]

[0799]

[0800]

[0801] on phaqe binding to

[0802]

[0803] The ability of BNP, a natural ligand of NPR3, to inhibit the binding of NPR3 binding Bicycle molecules displayed on phage to NPR3 was assessed by an Alphascreen competition assay.

[0804] NPR3 protein synthesis method

[0805] Codon optimized human NPR3 (Uniprot ID P17342) residues 46-485 with CD33 signal peptide and C-terminal TEV (Tobacco Etch Virus) protease site followed by 6xHis tag was cloned into an in-house pDEST12.2 derived mammalian expression vector (pEBNAZ). The protein was expressed by transient transfection of Expi293F cells in Expi293 expression medium (ThermoFisher Scientific) using PEI Max transfection reagent (PolySciences Inc.) at 1 :4 DNA: PEI Max ratio and 1.5 ug / ml DNA concentration. The cells were cultured for 6 days at 37°C, 8% CO2.

[0806] Supernatant containing secreted NPR3, supplemented with 20 mM HEPES pH 8.0 and 5 mM imidazole was loaded on to two 5 ml HiTrap Excel (Cytiva) columns on an Akta Pure (Cytiva) equilibrated with buffer A (20 mM HEPES pH 7.4, 500 mM NaCI, 20 mM imidazole), then washed with 5% buffer B (20 mM HEPES pH 7.4, 500 mM NaCI, 400 mM imidazole), until a stabil UV280nm baseline was obtained. The bound protein was then eluted with 100 % B and further purified by size exclusion chromatography using a Superdex 20026 / 60 column (Cytiva) in a buffer consisting of 20 mM HEPES pH 7.4, 200 mM NaCI and 10% glycerol. The appropriate peak was collected and concentrated to about 5 mg / ml and flash frozen in liquid nitrogen and stored at -80°C until required.

[0807] Sequence of Biotinylated NPR3 (Uniprot P17342:48-485) EALPPQKIEVLVLLPQDDSYLFSLTRVRPAIEYALRSVEGNGTGRRLLPPGTRFQVAYEDSD CGNRALFSLVDRVAAARGAKPDLILGPVCEYAAAPVARLASHWDLPMLSAGALAAGFQHK DSEYSHLTRVAPAYAKMGEMMLALFRHHHWSRAALVYSDDKLERNCYFTLEGVHEVFQEE GLHTSIYSFDETKDLDLEDIVRNIQASERWIMCASSDTIRSIMLVAHRHGMTSGDYAFFNIEL FNSSSYGDGSWKRGDKHDFEAKQAYSSLQTVTLLRTVKPEFEKFSMEVKSSVEKQGLNME DYVNMFVEGFHDAILLYVLALHEVLRAGYSKKDGGKIIQQTWNRTFEGIAGQVSIDANGDRY GDFSVIAMTDVEAGTQEVIGDYFGKEGRFEMRPNVKYPWGPLKLRIDENRIVEHTNSSPCK SSGGLEESAENLYFQSAAAHHHHHH (SEQ ID NO: 73)Biotinylated NPR3 (10nM) and representative phage clones presenting NPR3 binding Bicycle molecules were incubated with a titration of BNP (R&D Systems Cat. Number 3522 / 1 , 100nM-0.1pM, 1:10 dilutions) in assay buffer (25mM HEPES, 100mM NaCI, 0.5% BSA, 0.05% P20, pH7.4) for 30 minutes. 15ug / mL anti M13 Alphascreen acceptor beads were added (Perkin Elmer, Cat. Number 6762003 directly conjugated to antiM13 gene8 Antibody (VWR 27-9420-01) as per manufacturer’s instructions) and incubated for 30 minutes. Finally, 20ug / mL of Streptavidin Alphascreen donor beads (Perkin Elmer, Cat. Number 6760002S) was added and incubated for 60 minutes. Plates were read on a Pherastar FS / FSX (BMG Labtach) using excitation wavelength 680nm, emission wavelength 615nm. Data was normalised to beads alone (low) and no competitor (high) reference averages. Data was plotted in GraphPad Prism (version 10.1.0), as shown in Figure 2.

[0808] Bicycle molecules presented on phage clones demonstrating greater than 50% inhibition were identified as BNP competitors and therefore active site binders.

[0809] All Bicycle molecules presented on phage clones demonstrating a BNP concentration dependent inhibition of Alphascreen signal greater than 50% (denoted by the dotted line in Figure 2) are identified as active site binders as the phage clone:NPR3 Alphascreen binding signal has been inhibited by the presence of BNP, a known NPR3 active site ligand.

[0810] The percent inhibition was calculated for 100nM BNP samples using the formula: % inhibition = 100 - (normalised Alphascreen signal) and is shown in the table below. All Bicycle molecules presented on phage clones where the binding to NPR3 is inhibited by BNP by greater than 50% are identified as active site binders.

[0811] Table 3: Percent inhibition of NPR3 binding to phage clones displaying NPR3 binding Bicycle molecules by 100nM BNP, a known NPR3 active site ligand.

[0812]

[0813] Example 5: Inhibition of NPR3 binding Bicycle molecules presented on phage binding to biotinylated NPR3 by active site binder Bicycle molecule B8 by Alphascreen

[0814] The ability of an active site binding Bicycle molecule (B8), to inhibit the binding of NPR3 binding Bicycle molecules, displayed on phage to NPR3 was assessed by an Alphascreen competition assay. Biotinylated NPR3 (10nM) and representative phage clones presentingNPR3 binding Bicycle molecules were incubated with 10uM B8 (an active site binding Bicycle molecule) in assay buffer (25mM HEPES, 100mM NaCI, 0.5% BSA, 0.05% P20, pH7.4) for 30 minutes. 15ug / mL anti M13 Alphascreen acceptor beads were added (Perkin Elmer, Cat. Number 6762003 directly conjugated to antiM13 gene8 Antibody (VWR 27-9420-01) as per manufacturer’s instructions) and incubated for 30 minutes. Finally 20ug / mL of Streptavidin Alphascreen donor beads (Perkin Elmer, Cat. Number 6760002S) was added and incubated for 60 minutes. Plates were read on a Pherastar FS / FSX (BMG Labtach) using excitation wavelength 680nm, emission wavelength 615nm. Data was plotted in GraphPad Prism (version 10.1.0), as shown in Figure 3.

[0815] All Bicycle molecules presented on phage clones generating Alphascreen signals less than the non binding control are identified as active site binders as the binding signal has been inhibited by the presence of B8, a known active site binder.

[0816] 6: Inhibition of NPR3 binding Bicycle molecules presented on phaqe binding to biotinylated NPR3 by active site binder Bicycle molecule B8 by Alphascreen

[0817] The ability of and active site binding Bicycle molecule (B8), inhibit the binding of NPR3 binding Bicycle molecules, displayed on phage to NPR3 was assessed by an Alphascreen competition assay. Biotinylated NPR3 (10nM) and representative phage clones presenting NPR3 binding Bicycle molecules were incubated in the absence or presence of 10uM B8 (a known active site binding Bicycle molecule) in assay buffer (25mM HEPES, 100mM NaCI, 0.5% BSA, 0.05% P20, pH7.4)for30 minutes. 15ug / mL anti M13 Alphascreen acceptor beads were added (Perkin Elmer, Cat. Number 6762003 directly conjugated to antiM13 gene8 Antibody (VWR 27-9420-01 ) as per manufacturer’s instructions) and incubated for 30 minutes. Finally 20ug / mL of Streptavidin Alphascreen donor beads (Perkin Elmer, Cat. Number 6760002S) was added and incubated for 60 minutes. Plates were read on a Pherastar FS / FSX (BMG Labtach) using excitation wavelength 680nm, emission wavelength 615nm.

[0818] Percent inhibition of signal was calculated using the following formula:

[0819] ((Alphascreen signal generated in the presence of B8) / (Alphascreen signal generated in the absence ofB8))*100

[0820] Data was plotted in GraphPad Prism (version 10.1.0), as shown in Figure 4.

[0821] All Bicycle molecules presented on phage clones generating Alphascreen signals less than 50% maximum signal are identified as active site binders as the binding signal has been inhibited by the presence of B8, a known active site binder.

Claims

CLAIMS1. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, said peptide ligand comprising a polypeptide comprising at least three reactive groups, wherein the polypeptide is attached to a molecular scaffold.

2. The peptide ligand as defined in claim 1 , or a pharmaceutically acceptable salt thereof, wherein the three reactive groups of the polypeptide form covalent bonds with the molecular scaffold such that at least two polypeptide loops are formed on the molecular scaffold.

3. The peptide ligand or pharmaceutically acceptable salt thereof as defined in claim 1 or claim 2, wherein said peptide ligand binds the active site of NPR3.

4. The peptide ligand or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 3, said peptide ligand having the structure of formula (I):wherein Scaffold represents the molecular scaffold; andZ1-RG1-X1-RG2-X2-RG3-Z2 represents the polypeptide, wherein RG1, RG2 and RG3 represent the three reactive groups, Xi represents between 2 and 9 natural or non-natural amino acids which join with said RG1 and RG2 groups to form Loop 1 , X2 represents between 2 and 9 natural or non-natural amino acids which join with said RG2 and RG3 groups to form Loop 2, such that the total number of amino acid residues within Xi and X2 does not exceed 12, Z1 represents one or more optional N-terminal natural or non-natural amino acids or an acetyl group, andZ2 represents one or more optional C-terminal natural or non-natural amino acids.

5. The peptide ligand or pharmaceutically acceptable salt thereof as defined in claim 4, wherein Xi comprises an amino acid sequence selected from:VLPPWA (SEQ ID NO: 19);WW (SEQ ID NO: 20);RIDHL (SEQ ID NO: 21);LDEEV (SEQ ID NO: 22);ELWTHNSF (SEQ ID NO: 23);REFDLL (SEQ ID NO: 24);DWI (SEQ ID NO: 25);RIDPIVK (SEQ ID NO: 26);PLDRIG (SEQ ID NO: 27);AFG (SEQ ID NO: 28);KTL (SEQ ID NO: 29);RLDHSFM (SEQ ID NO: 30);PIDRLG (SEQ ID NO: 31);TFG (SEQ ID NO: 32);LSAWH (SEQ ID NO: 33);PLDRIS (SEQ ID NO: 34);HRMDKI (SEQ ID NO: 35); andNIDAIG (SEQ ID NO: 36).

6. The peptide ligand or pharmaceutically acceptable salt thereof as defined in claim 4 or claim 5, wherein X2 comprises an amino acid sequence selected from:AIY (SEQ ID NO: 37);YDTTLNL (SEQ ID NO: 38);GKYTL (SEQ ID NO: 39);LSYLLWG (SEQ ID NO: 40);IGMN (SEQ ID NO: 41);KPDWFS (SEQ ID NO: 42);AYLATKGL (SEQ ID NO: 43);PL (SEQ ID NO: 44);GRL (SEQ ID NO: 45);PLDRLSHY (SEQ ID NO: 46);FGGPLDHLV (SEQ ID NO: 47);DI (SEQ ID NO: 48);YSKNF (SEQ ID NO: 49);PIDPISI (SEQ ID NO: 50);PLDRIS (SEQ ID NO: 51);SSTF (SEQ ID NO: 52);MPSSF (SEQ ID NO: 53); andNPRF (SEQ ID NO: 54).

7. The peptide ligand or pharmaceutically acceptable salt thereof as defined in any one of claims 4 to 6, wherein RGi, RG2 and RG3 comprise cysteine residues.

8. The peptide ligand or pharmaceutically acceptable salt thereof as defined in any one of claims 4 to 7, wherein RG1-X1-RG2-X2-RG3 represents a polypeptide having an amino acid sequence having at least 70% sequence identity to any one of:CVLPPWACAIYC (SEQ ID NO: 1);CWWCYDTTLNLC (SEQ ID NO: 2);CRIDHLCGKYTLC (SEQ ID NO: 3);CLDEEVCLSYLLWGC (SEQ ID NO: 4);CELWTHNSFCIGMNC (SEQ ID NO: 5);CREFDLLCKPDWFSC (SEQ ID NO: 6);CDWICAYLATKGLC (SEQ ID NO: 7);CRIDPIVKCPLC (SEQ ID NO: 8);CPLDRIGCGRLC (SEQ ID NO: 9);CAFGCPLDRLSHYC (SEQ ID NO: 10);CKTLCFGGPLDHLVC (SEQ ID NO: 11);CRLDHSFMCDIC (SEQ ID NO: 12);CPIDRLGCYSKNFC (SEQ ID NO: 13);CTFGCPIDPISIC (SEQ ID NO: 14);CLSAWHCPLDRISC (SEQ ID NO: 15);CPLDRISCSSTFC (SEQ ID NO: 16);CHRMDKICMPSSFC (SEQ ID NO: 17); andCNIDAIGCNPRFC (SEQ ID NO: 18).

9. The peptide ligand or pharmaceutically acceptable salt thereof as defined in any one of claims 4 to 8, wherein RG1-X1-RG2-X2-RG3 represents a polypeptide having an amino acid sequence selected from:CVLPPWACAIYC (SEQ ID NO: 1);CWWCYDTTLNLC (SEQ ID NO: 2);CRIDHLCGKYTLC (SEQ ID NO: 3);CLDEEVCLSYLLWGC (SEQ ID NO: 4);CELWTHNSFCIGMNC (SEQ ID NO: 5);CREFDLLCKPDWFSC (SEQ ID NO: 6);CDWICAYLATKGLC (SEQ ID NO: 7);CRIDPIVKCPLC (SEQ ID NO: 8);CPLDRIGCGRLC (SEQ ID NO: 9);CAFGCPLDRLSHYC (SEQ ID NO: 10);CKTLCFGGPLDHLVC (SEQ ID NO: 11);CRLDHSFMCDIC (SEQ ID NO: 12);CPIDRLGCYSKNFC (SEQ ID NO: 13);CTFGCPIDPISIC (SEQ ID NO: 14);CLSAWHCPLDRISC (SEQ ID NO: 15);CPLDRISCSSTFC (SEQ ID NO: 16);CHRMDKICMPSSFC (SEQ ID NO: 17); andCNIDAIGCNPRFC (SEQ ID NO: 18).

10. The peptide ligand or pharmaceutically acceptable salt thereof as defined in any one of claims 4 to 9, wherein Zi represents an alanine residue or an acetyl group.

11. The peptide ligand or pharmaceutically acceptable salt thereof as defined in any one of claims 4 to 10, wherein Z2 is either absent or represents an alanine residue.

12. The peptide ligand or pharmaceutically acceptable salt thereof according to any one of claims 4 to 11 , wherein Z1-RG1-X1-RG2-X2-RG3-Z2 represents a polypeptide having an amino acid sequence selected from:A-(SEQ ID NO: 1)-A;A-(SEQ ID NO: 2)-A;A-(SEQ ID NO: 3)-A;A-(SEQ ID NO: 4)-A;A-(SEQ ID NO: 5)-A;A-(SEQ ID NO: 6)-A;A-(SEQ ID NO: 7)-A;A-(SEQ ID NO: 8)-A;A-(SEQ ID NO: 9)-A;Ac-(SEQ ID NO: 10);A-(SEQ ID NO: 11)-A;A-(SEQ ID NO: 12)-A;A-(SEQ ID NO: 13)-A;Ac-(SEQ ID NO: 14);A-(SEQ ID NO: 15)-A;A-(SEQ ID NO: 16)-A;A-(SEQ ID NO: 17)-A; andA-(SEQ ID NO: 18)-A.

13. The peptide ligand or pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the scaffold is a derivative of TATA which has the following structure:wherein * denotes the point of attachment of the three reactive groups, and the polypeptide additionally comprises N- and / or C-terminal additions and comprises an amino acid sequence which is selected from:A-(SEQ ID NO: 1)-A (herein referred to as B1);A-(SEQ ID NO: 2)-A (herein referred to as B2);A-(SEQ ID NO: 3)-A (herein referred to as B3);A-(SEQ ID NO: 4)-A (herein referred to as B4);A-(SEQ ID NO: 5)-A (herein referred to as B5);A-(SEQ ID NO: 6)-A (herein referred to as B6);A-(SEQ ID NO: 7)-A (herein referred to as B7);A-(SEQ ID NO: 8)-A (herein referred to as B8);A-(SEQ ID NO: 9)-A (herein referred to as B9);Ac-(SEQ ID NO: 10) (herein referred to as B10);A-(SEQ ID NO: 11)-A (herein referred to as B11);A-(SEQ ID NO: 12)-A (herein referred to as B12);A-(SEQ ID NO: 13)-A (herein referred to as B13);Ac-(SEQ ID NO: 14) (herein referred to as B14);A-(SEQ ID NO: 15)-A (herein referred to as B15);A-(SEQ ID NO: 16)-A (herein referred to as B16);A-(SEQ ID NO: 17)-A (herein referred to as B17); andA-(SEQ ID NO: 18)-A (herein referred to as B18).

14. A polypeptide, ora pharmaceutically acceptable salt thereof, comprising an amino acid sequence as defined in any one of claims 4 to 13.

15. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) B1.B-B-B-C-B-B-B2.B-B-B3.B-C,wherein B is any amino acid,Bi is C or Cam,B2 is D or E,B3 is A, D, S or Z-1 ; or(b) CTFGCPIDPISIC (SEQ ID NO: 14); or(c) CTFGCPIDPISIC (SEQ ID NO: 14) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

16. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-B1-B2-W-C-N-B3-B4-L-V-R-M-Y-Cwherein Bi is A or E or S,B2 is A or P or S or T,B3 is A L or M or P or S,B4 is H or M or N; or(b) CSSWCNSMLVRMYC; or(c) CSSWCNSMLVRMYC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

17. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CDWICAYLATKGLC (SEQ ID NO: 7).

18. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-B1-B2-B3-C-B4-L-D-B5-B6-B7-B8-B9-C,wherein Bi is A or K,B2 is F or 2Nal,B3 is G or K,B4 is P or Hyp,B5 is K or R,B6is I or L,B7 is S,Bs is A or H,Bg is V or Y;(b) CAFGCPLDRLSHYC (SEQ ID NO: 10); or(c) CAFGCPLDRLSHYC (SEQ ID NO: 10) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

19. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-B-B-B1.B2.B-C-B-B-B-B-B-B3,wherein B is any amino acid,Bi is AspT or Cya or D or tetrazole-Ala,B2 is Agb or H or NArg or Q,B3 is C or NMe C;(b) CRIDHLCGKYTLC (SEQ ID NO: 3); or(c) CRIDHLCGKYTLC (SEQ ID NO: 3) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

20. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CKTMIRCPYPGKC.

21. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CGRPSSCYANKFC.

22. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CGGENRCPTKKWC.

23. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-B1-L-P-B2-W-A-C-B3-I-Y-C,wherein Bi is A or D or E or N or T or V,B2 is D or I or L or P or Q or T or V,B3 is A or D or E or Q or V;(b) CVLPPWACAIYC (SEQ ID NO: 1); or(c) CVLPPWACAIYC (SEQ ID NO: 1) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

24. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-P-B1-D-R-I-G-C-B2-B3-B4-C,wherein Bi is F or L or M,B3 is G or R,B4 is R or L or S or T or M or N,B5 is A or F or L or M;(b) CPLDRIGCGRLC (SEQ ID NO: 9); or(c) CPLDRIGCGRLC (SEQ ID NO: 9) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

25. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPFDPIGCRHFC.

26. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-N-B1-L-L-B2-K-C-D-W-F-B3-C,wherein Bi is F or H or P or V or Y,B2 is A or S,B3 is N or Q or T;(b) CNPLLAKCDWFQC; or(c) CNPLLAKCDWFQC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

27. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-P-B1-D-B2-L-G-C-N-B3-B4-M-Cwherein Bi is I or M,B2 is R or P,B3 is L or P,B4 is R or G;(b) CPMDPLGCNLRMC; or(c) CPMDPLGCNLRMC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

28. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPLDRISCSSTFC (A-(SEQ ID NO: 16)-A).

29. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CNIDAIGCNPRFC (SEQ ID NO: 18).

30. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) B1.B-B-B2B-B3-B-B-C-B-B-B4,wherein B is any amino acid,Bi is C, meC, or [Im],B2 aMeD or D or tetrazole,B3 is Chg or EPA or F or I,B4 is C or meC;(b) CRIDPIVKCPLC (SEQ ID NO: 8); or(c) CRIDPIVKCPLC (SEQ ID NO: 8) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

31. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CRLDHSFMCDIC (A-(SEQ ID NO: 12)-A).

32. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-R-L-D-B1-B2-B3-B4-C-B5-L-C,wherein Bi is P or Q,B2 is I or L,B3 is T or S,B4is M or Q,B5 is K or N;(b) CRLDPLTQCKLC; or(c) CRLDPLTQCKLC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

33. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CKVDHVRMCGLC.

34. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CSYEELKRVCLMPC.

35. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptideattached to a molecular scaffold, and wherein the polypeptide comprises CDAWVCNYLRQKELC.

36. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-B1-B2-W-B3-C-B4-Y-B5-B6-B7-B8-B9-B10-C,wherein Bi is D or E,B2 is D or E or N or P or Q,B3is I or V,B4 is E or N or S or T,B5 is I or L or M,Be is H or N or R or S or T,B7 is A or H or L or S,Bs is K or M or R,Bg is G or H or L or M or N,B10 is L or M or N or S or V;(b) CDDWVCTYMSARGVC; or(c) CDDWVCTYMSARGVC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

37. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-W-W-C-Y-D-B1-B2-B3-B4-B5-C,wherein Bi or D or E or P or T,B2 is A or E or S or T,B3 is I or L or Q or V,B4 is N or D,B5 is I or L or M;(b) CWWCYDTTLNLC (SEQ ID NO: 2); or(c) CWWCYDTTLNLC (SEQ ID NO: 2) with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

38. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptideattached to a molecular scaffold, and wherein the polypeptide comprises CLDEEVCLSYLLWGC (SEQ ID NO: 4).

39. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CELWTHNSFCIGMNC (SEQ ID NO: 5).

40. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CREFDLLCKPDWFSC (SEQ ID NO: 6).

41. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CKTLCFGGPLDHLVC (SEQ ID NO: 11).

42. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises:(a) C-B1-P-F-B2-C-R-I-D-B3-B4-B2-B5-B6-C,wherein Bi is A or H or SB2 is G or SB3 is K or R or TB4 is I or LB5 is A or L or S or TBe is E or H or N or R;(b) CHPFSCRIDKLSAEC; or(c) CHPFSCRIDKLSAEC with five, four, three, two or one amino acid substitutions that do not add or remove a cysteine residue.

43. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPIDRLGCYSKNFC (SEQ ID NO: 13).

44. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CPKMDRICIIEGFC.

45. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CLSAWHCPLDRISC (A-(SEQ ID NO: 15)-A).

46. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CHRMDKICMPSSFC (SEQ ID NO: 17).

47. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises CRLDKLSCGRSSFC.

48. A peptide ligand capable of binding natriuretic peptide receptor 3 (NPR3), or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide attached to a molecular scaffold, and wherein the polypeptide comprises C-T-4FPhe-C-Hyp-tBuAla-D-P-tBuAla-S-l-C.

49. The peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 48, wherein the pharmaceutically acceptable salt is selected from the sodium, potassium, calcium, or ammonium salt.

50. The peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 49, wherein the NPR3 is human NPR3.

51. A pharmaceutical composition which comprises the peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 50, in combination with one or more pharmaceutically acceptable excipients.

52. The pharmaceutical composition as defined in claim 51, which additionally comprises one or more therapeutic agents.

53. A drug conjugate comprising the peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 50, conjugated to one or more effector and / or functional groups.

54. The peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 50, the pharmaceutical composition as defined in claim 51 or claim 52, or the drug conjugate as defined in claim 53, for use in therapy.

55. The peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 50, the pharmaceutical composition as defined in claim 51 or claim 52, or the drug conjugate as defined in claim 53, for use in preventing, suppressing, or treating a disease or disorder mediated by the natriuretic peptide (NP) system.

56. The peptide ligand, polypeptide, or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 50, the pharmaceutical composition as defined in claim 51 or claim 52, or the drug conjugate as defined in claim 53, for use in preventing, suppressing, or treating renal disease.

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