Bicyclic peptide ligands that bind to PBP

Abstract

The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide loops are present oppositely between the attachment points of the scaffold. In particular, the present invention describes peptides that are high-affinity binders of penicillin binding proteins (PBPs). The present invention also includes pharmaceutical compositions comprising the peptide ligands, and the use of the peptide ligands in inhibiting or treating diseases or disorders mediated by bacterial infection, or in providing prevention to subjects at risk of infection.

Description

Technical Field

[0001] The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide loops subtend between the attachment points of the scaffold. In particular, the present invention describes peptides that are high-affinity binders of penicillin binding proteins (PBPs). The present invention also includes pharmaceutical compositions comprising the peptide ligands, and the use of the peptide ligands in inhibiting or treating diseases or disorders mediated by bacterial infection, or in providing prevention to subjects at risk of infection. Background Art

[0002] Cyclic peptides can bind to protein targets with high affinity and target specificity, and are therefore an attractive class of molecules for therapeutic agent development. In fact, several cyclic peptides have been successfully used clinically, such as the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of hundreds of square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 ( Wu et al. (2007), Science 330, 1066-71), with integrin αVb3 The cyclic peptides with Arg-Gly-Asp motifs (Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 ( Zhao et al. (2007), J Struct Biol 160(1), 1-10).

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss upon binding to the target and resulting in higher binding affinity. Reduced flexibility also results in locking of 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) that loses selectivity relative to other MMPs upon ring opening (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties obtained by macrocyclization are more pronounced in polycyclic peptides having more than one peptide ring, such as in vancomycin, nisin, and actinomycin.

[0004] Different research groups have previously tethered peptides with cysteine ​​residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers have used tri(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings to synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). WO 2004 / 077062 and WO 2006 / 078161 disclose methods for generating candidate drug compounds, wherein the compounds are generated by attaching cysteine-containing peptides to a molecular scaffold, such as tri(bromomethyl)benzene.

[0005] Combinatorial methods based on phage display have been developed to generate and screen large libraries of bicyclic peptides against target targets (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine ​​residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chains to a small molecule scaffold. Summary of the invention

[0006] According to a first aspect of the present invention, a peptide ligand capable of binding to one or more penicillin-binding proteins (PBPs) is provided, which comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two ring sequences, and the molecular scaffold forms a covalent bond with the cysteine ​​residues of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold.

[0007] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand as defined herein, in combination with one or more pharmaceutically acceptable excipients.

[0008] According to a further aspect of the present invention there is provided a peptide ligand as defined herein for use in inhibiting or treating a disease or condition mediated by a bacterial infection, or for providing prophylaxis to a subject at risk of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1: Direct binding data from fluorescence polarization assays of BCY12130 binding to PBP3 from A. baumannii (circles), E. coli (triangles), and P. aeruginosa (squares).

[0010] Figure 2 : A) Competition binding data of the E. coli PBP3 fluorescence polarization assay using BCY12130 competing with Bocillin (BODIPY-penicillin). B) Competition binding data of the E. coli PBP3 fluorescence polarization assay as described in A, but using BCY12130 (circles, Ki 0.24 μM) and carbenicillin (squares, Ki 0.52 μM).

[0011] Figure 3 : A) Microscopic images showing the morphology of E. coli grown in the absence or presence of the indicated concentrations of BCY12130. B) As in A, but for S. typhimurium. C) As in B, but for E. cloacae (all scale bars = 10 μm). DETAILED DESCRIPTION

[0012] In one embodiment, the loop sequence comprises 2, 3, 4, 5, 6, 7, 8 or 9 amino acids.

[0013] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, each consisting of 4 amino acids. Examples of such loop sequences are those within BCY12132 as described herein.

[0014] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 4 amino acids and the other of which consists of 5 amino acids. Examples of such loop sequences are those within BCY9377, BCY9378, BCY12130, BCY10020, BCY10022, BCY10024, BCY10025 and BCY10026 as described herein.

[0015] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 8 amino acids and the other of which consists of 2 amino acids. Examples of such loop sequences are those within BCY9381, BCY9226 and BCY9229 as described herein.

[0016] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 8 amino acids and the other of which consists of 3 amino acids. Examples of such loop sequences are those within BCY9382, BCY9383, BCY9389, BCY9391, BCY10027 and BCY10028 as described herein.

[0017] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 5 amino acids and the other of which consists of 7 amino acids. Examples of such loop sequences are those within BCY9384 and BCY9385 as described herein.

[0018] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 5 amino acids and the other of which consists of 5 amino acids. Examples of such loop sequences are those within BCY9386, BCY13797, BCY14381, BCY14613, BCY14618, BCY14619, BCY14621, BCY14627, BCY14629, BCY14631 and BCY14641 as described herein.

[0019] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 3 amino acids and the other of which consists of 7 amino acids. Examples of such loop sequences are those within BCY9387 as described herein.

[0020] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 2 amino acids and the other of which consists of 7 amino acids. Examples of such loop sequences are those within BCY9388 as described herein.

[0021] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 4 amino acids and the other of which consists of 8 amino acids. Examples of such loop sequences are those within BCY9227 and BCY9233 as described herein.

[0022] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 3 amino acids and the other of which consists of 9 amino acids. Examples of such loop sequences are those within BCY9237 as described herein.

[0023] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of 6 amino acids and the other consists of 6 amino acids. Examples of such loop sequences are those within BCY9238 as described herein.

[0024] The PBPs referred to herein include "penicillin binding proteins," which may be present in any bacterial species. In one embodiment, the PBP is a PBP present in one or more pathogenic bacterial species. In a further embodiment, the one or more pathogenic bacterial species are selected from any of the following: Acinetobacter baumannii, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, tetani), Corynebacterium diphtheriae, Echinococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (such as enterotoxigenic, enteropathogenic, enterohemorrhagic, or enteroaggregative), Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosistuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pneumococcus, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella (such as Salmonella bongori, Salmonella enterica, Salmonella subterranean, Salmonella typhi, or Salmonella typhimurium), Shigella (such as Shigella sonnei or Shigella dysenteriae), Staphylococcus aureus aureus (such as MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibriocholerae, or Yersinia pestis.

[0025] In one embodiment, the PBP is a PBP present in Streptococcus pneumoniae. In a further embodiment, the PBP present in Streptococcus pneumoniae is selected from the following five PBPs: 1a, 1b, 2a, 2x and 2b. In a further embodiment, the PBP present in Streptococcus pneumoniae is PBP1a.

[0026] In an optional embodiment, the PBP is a PBP present in E. coli. In a further embodiment, the PBP present in E. coli is selected from the following 12 PBPs: 1a, 1b, 1c, 2, 3, 4, 5, 6, 7 / 8, DacD, AmpC and AmpH. In a further embodiment, the PBP present in E. coli is PBP1b. In another embodiment, the PBP present in E. coli is PBP3.

[0027] In a further embodiment, the PBP is a PBP present in Pseudomonas aeruginosa. In an even further embodiment, the PBP present in Pseudomonas aeruginosa is selected from the following 7 PBPs: 1a, 1b, 2, 3, 3a, 4 and 5. In a further embodiment, the PBP present in Pseudomonas aeruginosa is PBP3.

[0028] In a further embodiment, the PBP is a PBP present in Acinetobacter baumannii. In an even further embodiment, the PBP present in Acinetobacter baumannii is selected from the following 9 PBPs: 1a, 1b, 2, 3, 4, 5, 6, 7 and 8. In a further embodiment, the PBP present in Acinetobacter baumannii is PBP3.

[0029] In one embodiment, cell division requires a PBP, such as FtsI. In a further embodiment, the FtsI is present in Escherichia coli, Acinetobacter baumannii or Pseudomonas aeruginosa and is referred to as PBP3. Thus, according to certain embodiments of the present invention, PBP3 is FtsI. In one embodiment, the PBP is not PBP3 and / or Fts1.

[0030] In one embodiment, the PBP is S. pneumoniae PBP1a and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0031] C i RFSSC ii PPYHVC iii (SEQ ID NO: 1);

[0032] C i PYTSC ii PPHTMC iii (SEQ ID NO: 2);

[0033] C i HPRHQEGYC ii MPC iii (SEQ ID NO: 3);

[0034] C iYNHKWGAMC ii THPC iii (SEQ ID NO:4):

[0035] C i HDWDYRHLC ii YWRC iii (SEQ ID NO:5):

[0036] C i DIYREC ii HYTSWSVC iii (SEQ ID NO:6):

[0037] C i KPSLSC ii QHLPRALC iii (SEQ ID NO:7):

[0038] C i PFTGPC ii RPHYIC iii (SEQ ID NO:8):

[0039] C i YTSC ii PEHHVFAC iii (SEQ ID NO:9):

[0040] C i DNC ii PAPER iii (SEQ ID NO:10):

[0041] C i NPRC ii HPVYTSFFC iii (SEQ ID NO:11):

[0042] C i GAPC ii RPHYVPWFC iii (SEQ ID NO:12):

[0043] C i PPVC ii RPHYVHWMC iii (SEQ ID NO:21):

[0044] C i PVGC ii RPHYVHWSC iii (SEQ ID NO:22):

[0045] C i RYTSC ii PPYTVC iii (SEQ ID NO: 23);

[0046] C i PYTSC ii PPYTHC iii (SEQ ID NO: 24);

[0047] C i PYTTC ii PPYHAC iii (SEQ ID NO: 25);

[0048] C i VFTTC ii PPYTVC iii (SEQ ID NO: 26); and

[0049] C i TYTTC ii PPFTIC iii (SEQ ID NO: 27),

[0050] or a pharmaceutically acceptable salt thereof, wherein C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively.

[0051] In a further embodiment, the PBP is S. pneumoniae PBP1a and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0052] A-(SEQ ID NO: 1)-A(BCY9377);

[0053] A-(SEQ ID NO: 2)-A(BCY9378);

[0054] A-(SEQ ID NO: 3)-A(BCY9381);

[0055] A-(SEQ ID NO: 4)-A(BCY9382);

[0056] A-(SEQ ID NO: 5)-A(BCY9383);

[0057] A-(SEQ ID NO: 6)-A(BCY9384);

[0058] A-(SEQ ID NO: 7)-A(BCY9385);

[0059] A-(SEQ ID NO: 8)-A(BCY9386);

[0060] A-(SEQ ID NO: 9)-A(BCY9387);

[0061] A-(SEQ ID NO: 10)-A(BCY9388);

[0062] A-(SEQ ID NO: 11)-A(BCY9389);

[0063] A-(SEQ ID NO: 12)-A(BCY9391);

[0064] A-(SEQ ID NO: 21)-A(BCY10028);

[0065] A-(SEQ ID NO:22)-A(BCY10027);

[0066] A-(SEQ ID NO: 23)-A(BCY10026);

[0067] A-(SEQ ID NO: 24)-A(BCY10025);

[0068] A-(SEQ ID NO: 25)-A(BCY10024);

[0069] A-(SEQ ID NO: 26)-A(BCY10022); and

[0070] A-(SEQ ID NO: 27)-A(BCY10020),

[0071] or a pharmaceutically acceptable salt thereof.

[0072] In one embodiment, the PBP is E. coli PBP1b and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0073] C i VYAPENLLC ii GSC iii (SEQ ID NO: 13);

[0074] C i SNPTC ii VYTPTNLFC iii (SEQ ID NO: 14);

[0075] C i NTC iiIYASENLLC iii (SEQ ID NO:15);

[0076] C i SATWGSRSC ii PVKFC iii (SEQ ID NO:16);

[0077] C i PNAC ii WTVHYSGYQC iii (SEQ ID NO:17);

[0078] C i HEFSLDC ii ILFGTSC iii (SEQ ID NO:18);

[0079] C i WGSWRC ii PIVHSC iii (SEQ ID NO:28);

[0080] C i WGSLRC ii PIVHSC iii (SEQ ID NO:29);

[0081] C i WGSLRC ii PIHYSC iii (SEQ ID NO:30);

[0082] C i WGSLRC ii PIKWDC iii (SEQ ID NO:31);

[0083] C i WGSLRC ii PITAHC iii (SEQ ID NO:32);

[0084] C i WGSKAC ii PITWHC iii (SEQ ID NO:33);

[0085] C i WGSRQC ii PISWTC iii (SEQ ID NO:34);

[0086] Ci QUR ii PVG iii (SEQ ID NO: 35);

[0087] C i WGSKSC ii PITWKC iii (SEQ ID NO:36); and

[0088] C i WGTSAC ii PVTHEC iii (SEQ ID NO: 37),

[0089] or a pharmaceutically acceptable salt thereof, wherein C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively.

[0090] In a further embodiment, the PBP is E. coli PBP1b and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0091] A-(SEQ ID NO: 13)-A(BCY9226);

[0092] A-(SEQ ID NO: 14)-A(BCY9227);

[0093] A-(SEQ ID NO: 15)-A(BCY9229);

[0094] A-(SEQ ID NO: 16)-A(BCY9233);

[0095] A-(SEQ ID NO: 17)-A(BCY9237);

[0096] A-(SEQ ID NO: 18)-A(BCY9238);

[0097] A-(SEQ ID NO: 28)-A(BCY14613);

[0098] A-(SEQ ID NO: 29)-A(BCY13797);

[0099] A-(SEQ ID NO: 30)-A(BCY14618);

[0100] A-(SEQ ID NO: 31)-A(BCY14621);

[0101] A-(SEQ ID NO: 32)-A(BCY14619);

[0102] A-(SEQ ID NO: 33)-A(BCY14627);

[0103] A-(SEQ ID NO: 34)-A(BCY14629);

[0104] A-(SEQ ID NO: 35)-A(BCY14631);

[0105] A-(SEQ ID NO:36)-A(BCY14641); and

[0106] A-(SEQ ID NO: 37)-A(BCY14381),

[0107] or a pharmaceutically acceptable salt thereof.

[0108] In one embodiment, the PBP is E. coli PBP3 and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0109] C i SFPKC ii PWV iii (SEQ ID NO: 19); and

[0110] C i RTFGC ii WWEGC iii (SEQ ID NO: 20),

[0111] or a pharmaceutically acceptable salt thereof, wherein C i , C ii and C iii The first, second and third cysteine ​​residues are indicated.

[0112] In a further embodiment, the PBP is E. coli PBP3 and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0113] A-(SEQ ID NO: 19)-A (referred to herein as BCY12130); and

[0114] A-(SEQ ID NO: 20)-A (referred to herein as BCY12132),

[0115] or a pharmaceutically acceptable salt thereof.

[0116] In one embodiment, the peptide ligand is not BCY12130 and BCY12132.

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th edition, John Wiley & Sons, Inc.), which are incorporated herein by reference.

[0118] the term

[0119] serial number

[0120] When referring to amino acid residue positions within the peptides of the invention, cysteine ​​residues (C i , C ii and C iii ) is unchanged and omitted from the numbering, therefore, the numbering of the amino acid residues in the peptides of the present invention is mentioned as follows:

[0121] C i -R1-F2-S3-S4-C ii -P5-P6-Y7-H8-V9-C iii (SEQ ID NO: 1).

[0122] For the purpose of this description, it is assumed that all bicyclic peptides cyclize with 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) to produce a trisubstituted structure. Cyclization with TATA occurs at C i , C ii and C iii superior.

[0123] Molecular form

[0124] The N- or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence, separated by a hyphen. For example, the N-terminal βAla-Sar10-Ala tail would be represented as:

[0125] βAla-Sar10-A-(SEQ ID NO:X).

[0126] Reverse peptide sequence

[0127] According to the disclosure of Nair et al. (2003), J Immunol 170(3), 1362-1373, it is contemplated that the peptide sequences disclosed herein will also be used in their retro-inverse form. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa), and its stereochemistry is also reversed (i.e., D-amino acids become L-amino acids, and vice versa).

[0128] Peptide ligand

[0129] As referred to herein, a peptide ligand refers to a peptide that is covalently bound to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) that are capable of forming a covalent bond with the scaffold, and a sequence that is present opposite the reactive groups, which is referred to as a loop sequence because it forms a loop when the peptide is bound to the scaffold. In this case, the peptide comprises at least three cysteine ​​residues (referred to herein as C i , C ii and C iii ), and forming at least two rings on the bracket.

[0130] Advantages of peptide ligands

[0131] Certain bicyclic peptides of the present invention have many favorable properties that make them considered as drug-like molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. Such favorable properties include:

[0132] - Species cross-reactivity. Certain ligands exhibit cross-reactivity between PBPs from different bacterial species, thus being able to treat infections caused by multiple bacterial species. Other ligands may be highly specific for PBPs from certain bacterial species, which may be beneficial in treating infections without causing collateral damage to the patient's beneficial flora;

[0133] - Protease stability. Bicyclic peptide ligands should ideally exhibit stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across species so that bicyclic lead candidates can be developed in animal models and administered to humans with confidence;

[0134] - Ideal solubility profile. This is a function of the ratio of charged and hydrophilic residues to hydrophobic residues and intra / inter-molecular hydrogen bonding, which is important for formulation and absorption purposes;

[0135] - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides with short exposure in the acute disease management setting; or to develop bicyclic peptides with enhanced retention in the circulation, which are therefore optimal for treating more chronic disease states. Other factors contributing to the ideal plasma half-life are the requirement for sustained exposure to achieve maximum therapeutic efficacy, versus the toxicology that accompanies sustained exposure to the agent; and

[0136] - Selectivity. Certain peptide ligands of the invention exhibit selectivity for specific PBP isoforms, and certain other peptide ligands of the invention may inhibit more than one PBP isoform.

[0137] Pharmaceutically acceptable salts

[0138] It will be appreciated that salt forms are within the scope of the present invention and reference to a peptide ligand includes salt forms of the ligand.

[0139] The salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), 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 a suitable base or acid in water or in an organic solvent, or in a mixture of the two.

[0140] Acid addition salts (mono or di) can be formed with a wide variety of inorganic and organic acids. Examples of acid addition salts include mono or di salts formed with an acid selected from acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphor, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid , hippuric acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0141] A particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthylsulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One particular salt is the hydrochloride. Another particular salt is the acetate.

[0142] If the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be -COO - ), then it can form a salt with an organic or inorganic base to generate 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 Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g. NH3R+ NH2R2 + 、NHR3 + and NR4 + ). Some examples of 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 + .

[0143] When the peptide of the present invention comprises an amine functional group, it can be reacted with an alkylating agent to form a quaternary ammonium salt, for example, according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptide of the present invention.

[0144] Modified derivatives

[0145] It will be understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues (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 spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; replacement of one or more amino acid residues with alanine, replacement of one or more D-amino acids with succinimide; replacement of one or more amino acid residues ... The invention also provides methods for replacing one or more L-amino acid residues with a residue; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; modification of the length of the peptide backbone; replacing the hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modifying amino acids (such as cysteine, lysine, glutamate / aspartate and tyrosine) with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introducing or replacing amino acids with orthogonal reactivity suitable for functionalization, such as amino acids with azide or alkynyl groups, which allow functionalization with moieties with alkynyl or azido groups, respectively.

[0146] 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 a suitable amino-reactive chemistry and / or a C-terminal modification using a suitable carboxyl-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radiation chelator, or a chromophore.

[0147] 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, during peptide synthesis, the N-terminal cysteine ​​group (referred to herein as C i The group) is blocked by acetic anhydride or other suitable reagents, resulting in the molecule being acetylated at the N-terminus. This embodiment provides the advantage of removing potential recognition points of aminopeptidases and avoids the possibility of degradation of the bicyclic peptide.

[0148] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of an effector group and maintains the potency of the bicyclic peptide towards its target.

[0149] 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, during peptide synthesis, the C-terminal cysteine ​​group (referred to herein as C iii The group) is synthesized as an amide, resulting in a C-terminal amidation of the molecule. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidases and reduces the likelihood of proteolytic degradation of the bicyclic peptide.

[0150] In one embodiment, the modified derivatives include replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids with isosteric / isoelectronic side chains can be selected that are neither recognized by degradative proteases nor have any adverse effects on target efficacy.

[0151] Alternatively, unnatural amino acids with constrained amino acid side chains can be used so that proteolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, this involves proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid (Aib)), and cyclic amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

[0152] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises an N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii ) to add a spacer group.

[0153] In one embodiment, the modified derivative comprises replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues.

[0154] In one embodiment, the modified derivatives include replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an optional embodiment, the modified derivatives include replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged and hydrophobic amino acid residues is an important feature of the bicyclic peptide ligand. For example, hydrophobic amino acid residues affect the degree of plasma protein binding, thereby affecting the concentration of the free available part in the plasma, and charged amino acid residues (particularly arginine) can affect the interaction of the peptide with the cell surface phospholipid membrane. The two are combined to affect the half-life, distribution volume and exposure of the peptide drug, and can be adjusted according to the clinical endpoint. In addition, (if the peptide drug has been subcutaneously administered) the correct combination and quantity of charged and hydrophobic amino acid residues can reduce the stimulation at the injection site.

[0155] In one embodiment, the modified derivative comprises replacing 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 the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005), PNAS, 102 (2), 413-418).

[0156] In one embodiment, the modified derivatives include removal of any amino acid residues and substitution with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

[0157] It should be noted that each of the above modifications is used to intentionally improve the potency or stability of the peptide. Through modification, the potency can be further improved by the following mechanisms:

[0158] - Incorporation of hydrophobic moieties that exploit hydrophobic interactions and result in lower off-rates, allowing higher affinity to be achieved;

[0159] - incorporation of charged groups that exploit long-range ionic interactions, resulting in faster association rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0160] - Incorporating additional constraints into the peptide, for example by properly constraining the amino acid side chains to minimize entropy loss upon target binding, by restricting the torsion angles of the backbone to minimize entropy loss upon target binding, and introducing additional cyclization into the molecule for the same reason.

[0161] (For review, see Gentilucci et al., Curr. Pharmaceutical Design (2010) 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009) 16, 4399-418).

[0162] Isotopic variants

[0163] The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present invention, in which 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 occurring in nature, and peptide ligands of the present invention, in which a metal chelating group (referred to as "effector") is attached, which is capable of holding the relevant (radio)isotope, and peptide ligands of the present invention, in which certain functional groups are covalently substituted by relevant (radio)isotopes or isotopically labeled functional groups.

[0164] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include hydrogen isotopes, such as 2 H(D) and 3 H(T); Carbon isotopes such as 11 C. 13 C and 14 C; chlorine isotopes such as 36 Cl; fluorine isotopes such as 18 F; iodine isotopes such as 123 I. 125 I and 131 I; Nitrogen isotopes such as 13 N and 15 N; oxygen isotopes such as 15 O. 17 O and 18 O; phosphorus isotopes such as 32 P; sulfur isotopes such as 35 S; copper isotopes such as 64 Cu; Gallium isotopes such as 67 Ga or 68 Ga; yttrium isotopes such as 90 Y; and lutetium isotopes such as 177 Lu; and bismuth isotopes such as 213 Bi.

[0165] Certain isotope-labeled peptide ligands of the present invention, such as those incorporating radioactive isotopes, can be used for tissue distribution studies of drugs and / or substrates. The peptide ligands of the present invention can further have valuable diagnostic properties, which can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with labeling agents, such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, fluorescein, aequorin and luciferase), etc. The radioactive isotope tritium, i.e. 3 H(T) and carbon-14 14 C, is particularly useful for this purpose due to its ease of incorporation and readily available detection methods.

[0166] Using heavier isotopes such as deuterium 2 H(D) substitution may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0167] Positron emitting isotopes such as 11 C. 18 F. 15 O and 13 N substitution can be used in positron emission tomography (PET) studies to examine target occupancy.

[0168] Isotopically-labeled compounds of the peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0169] Molecular scaffold

[0170] In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. "Non-aromatic molecular scaffold" referred to herein refers to any molecular scaffold as defined herein that does not contain an aromatic (ie, unsaturated) carbocyclic or heterocyclic ring system.

[0171] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014), Angewandte Chemie, International Edition 53(6), 1602-1606.

[0172] As mentioned in the above document, the molecular scaffold may be a small molecule, such as an organic small molecule.

[0173] 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.

[0174] In one embodiment, the molecular scaffold comprises reactive groups capable of reacting with functional groups of a polypeptide to form covalent bonds.

[0175] The molecular scaffold may comprise chemical groups that form linkages with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0176] An example of a compound containing an αβ unsaturated carbonyl group is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie International Edition (2014), 53(6), 1602-1606).

[0177] synthesis

[0178] The peptides of the present invention can be synthesized by standard techniques and then reacted in vitro with the molecular scaffold. When doing this, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or verification. Such methods can be accomplished using conventional chemical methods as disclosed in Timmerman et al. (supra).

[0179] Therefore, the present invention also relates to the production of a polypeptide selected as described herein, wherein said production comprises optional further steps as described below. In one embodiment, these steps are performed on a final product polypeptide prepared by chemical synthesis.

[0180] The peptide may also be extended to incorporate, for example, another loop and thereby introduce multiple specificities.

[0181] To extend the peptide, conventional solid phase or solution phase chemistry can be used, using orthogonally protected lysine (and analogs) to simply chemically extend at its N-terminus or C-terminus or within a loop. Standard (bio) conjugation techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, addition can be made by fragment condensation or native chemical ligation, such as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266: 776-779), or by enzyme addition, such as using a subtiligase as described in (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91(26): 12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, 6000-6003 pages).

[0182] Alternatively, the peptide may be extended or modified by further conjugation of disulfide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) may be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; further cysteine ​​or thiol may then be attached to the N- or C-terminus of the first peptide so that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0183] Similar techniques are also used for the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[0184] Furthermore, other functional groups or effector groups can be added in the same manner, at the N- or C-terminus or via side chain coupling using appropriate chemistry. In one embodiment, the coupling is performed in a manner that does not block the activity of either entity.

[0185] Pharmaceutical composition

[0186] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand as defined herein, in combination with one or more pharmaceutically acceptable excipients.

[0187] Generally, the peptide ligands of the present invention will be used in purified form with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / water solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride and lactated Ringer's solution. If it is necessary to keep the polypeptide complex suspended, a suitable physiologically acceptable adjuvant can be selected from thickeners such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin and alginate.

[0188] 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).

[0189] The compounds of the invention can be used alone or in combination with another agent or agents. The other agent used in combination can be, for example, another antibiotic, or an antibiotic "adjuvant", such as an agent used to increase the permeability of gram-negative bacteria, a resistance determinant inhibitor, or a virulence mechanism inhibitor.

[0190] Suitable antibiotics for use in combination with the compounds of the present invention include, but are not limited to:

[0191] β-lactams, such as penicillins, cephalosporins, carbapenems or monobactams. Suitable penicillins include oxacillin, methicillin, ampicillin, cloxacillin, carbenicillin, piperacillin, ticarcillin, flucloxacillin and nafcillin; suitable cephalosporins include cefazolin, cephalexin, cephalothin, ceftazidime, cefepime, ceftobiprole, ceftaroline, ceftolozane and cefiderocol; suitable carbapenems include meropenem, doripenem, imipenem, ertapenem, biapenem and tebipenem; suitable monobactams include aztreonam;

[0192] Lincosamides, such as clindamycin and lincomycin;

[0193] Macrolides, such as azithromycin, clarithromycin, erythromycin, telithromycin, and solithromycin;

[0194] Tetracyclines, such as tigecycline, omadacycline, eravacycline, doxycycline, and minocycline;

[0195] Quinolones, such as ciprofloxacin, levofloxacin, moxifloxacin, and delafloxacin;

[0196] rifamycins, such as rifampin, rifabutin, rifalazil, rifapentine, and rifaximin;

[0197] Aminoglycosides, such as gentamicin, streptomycin, tobramycin, amikacin, and plazomicin;

[0198] Glycopeptides, such as vancomycin, teichoplanin, telavancin, dalbavancin, and oritavancin;

[0199] Pleuromutilins, such as lefamulin;

[0200] Oxazolidinones, such as linezolid or tedizolid;

[0201] Polymyxins, such as polymyxin B or colistin;

[0202] Trimethoprim, iclaprim, sulfamethoxazole;

[0203] Metronidazole;

[0204] Fidaxomicin:

[0205] Mupirocin;

[0206] Fusidic acid;

[0207] Daptomycin;

[0208] Murepavidin;

[0209] Fosfomycin; and

[0210] Nitrofurantoin.

[0211] Suitable antibiotic "adjuvants" include, but are not limited to:

[0212] Drugs known to improve bacterial uptake, such as outer membrane permeabilizing agents or efflux pump inhibitors; outer membrane permeabilizing agents may include polymyxin B nonapeptide or other polymyxin analogs, or edetate sodium;

[0213] Inhibitors of resistance mechanisms, such as beta-lactamase inhibitors; suitable beta-lactamase inhibitors include clavulanic acid, tazobactam, sulbactam, avibactam, relebactam, and nacubactam; and

[0214] Inhibitors of virulence mechanisms (e.g., toxins and secretion systems), including antibodies.

[0215] The compounds of the invention may also be used in combination with biological therapies, such as nucleic acid-based therapies, antibodies, bacteriophages or bacteriophage lytic enzymes.

[0216] The route of administration of the pharmaceutical composition according to the present invention can be any route generally known to those of ordinary skill in the art. For treatment, the peptide ligand of the present invention can be administered to any patient according to standard techniques. The route of administration includes, but is not limited to: oral (e.g., by ingestion); buccal; sublingual; transdermal (including, for example, by patch, plaster, etc.); transmucosal (including, for example, by patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy, such as by using an aerosol, such as oral or nasal); rectal (e.g., suppository or enema); vaginal (e.g., by vaginal suppository); parenteral, such as by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcutaneous, intraarticular, subarachnoid and substernal injection; by implanting a depot or reservoir, such as subcutaneous or intramuscular implantation. Preferably, the pharmaceutical composition according to the present 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, contraindications and other parameters to be considered by the clinician.

[0217] The peptide ligands of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and lyophilization and reconstitution techniques known in the art may be employed. Those skilled in the art will recognize that lyophilization and reconstitution may result in varying degrees of loss of activity and that levels may have to be adjusted upwards to compensate.

[0218] Compositions comprising the peptide ligands of the present invention or mixtures thereof can be administered for therapeutic treatment. In certain therapeutic applications, an amount sufficient to complete at least partial inhibition, suppression, regulation, killing or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease and the general state of the patient's own immune system, but is generally 10 μg to 250 mg of the selected peptide ligand per kilogram of body weight, with a more commonly used dose of 100 μg to 25 mg / kg / dose.

[0219] Compositions comprising peptide ligands according to the present invention can be used in therapeutic settings to treat microbial infections or to provide prevention for subjects at risk of infection (e.g., undergoing surgery, chemotherapy, artificial ventilation, or other conditions or planned interventions). In addition, peptide ligands as described herein can be selectively used to kill, consume, or otherwise effectively remove target cell populations from heterogeneous cell collections in vitro (extracorporeally) or in vitro (in vitro). Blood from mammals can be combined in vitro with selected peptide ligands, thereby killing or otherwise removing undesirable cells from blood for return to mammals according to standard techniques.

[0220] Therapeutic Uses

[0221] The bicyclic peptides of the present invention have particular utility as PBP binding agents.

[0222] Penicillin binding proteins (PBPs) are a group of proteins characterized by their affinity and binding ability to penicillin, which are present in many bacterial species. All β-lactam antibiotics (except tabtoxinine-β-lactam, which inhibits glutamine synthetase) bind to PBPs necessary for bacterial cell wall synthesis. PBPs are members of a subgroup of enzymes called transpeptidases. Specifically, some PBPs are DD-transpeptidases, while bifunctional PBPs have transglycosylase activity. PBPs are all involved in the final stages of peptidoglycan synthesis, which is the main component of bacterial cell walls. Bacterial cell wall synthesis is essential for bacterial growth, cell division (and thus reproduction) and maintenance of cell structure. Inhibition of PBPs leads to irregularities in cell wall structure, such as elongation, damage, loss of selective permeability, and ultimately cell death and lysis. Macheboeuf et al. (2006) FEMS Microbiology Reviews 30 (5), 673-691 provide a review of PBPs.

[0223] Therefore, without being bound by theory, it is believed that the peptide ligands of the present invention will be able to cause bacterial growth inhibition, cell death and lysis by binding to PBP and inhibiting cell wall synthesis. Silver (2007) Nature Reviews Drug Discovery 6, 41-55 and Zervosen et al. (2012) Molecules 17 (11), 12478-12505 provide a review of PBP as a therapeutic target. It will be understood that the peptide ligands of the present invention can bind to PBP at any site that can interfere with the mechanism of action of the PBP. For example, the peptide ligand can bind to the active site of the PBP and inhibit transpeptidase or transglycosylase. Alternatively, the peptide ligand can bind to other positions on the PBP to interfere with its mechanism of action.

[0224] The polypeptide ligands selected according to the methods of the present invention can be used for in vivo therapeutic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, etc. In certain applications such as vaccine applications, the ability to induce an immune response to a predetermined range of antigens can be used to adjust the vaccine to specific diseases and pathogens.

[0225] Substantially pure peptide ligands having at least 90% to 95% homogeneity are preferred for administration to mammals, and for pharmaceutical use, particularly when the mammal is a human, 98% to 99% or higher homogeneity is most preferred. The selected polypeptide, once partially purified or purified to the desired homogeneity, can be used for diagnosis or therapy (including in vitro) or for developing and conducting experimental procedures, immunofluorescence staining, etc. (Lefkovite and Pernis (1979 and 1981), Immunological Methods, Volumes I and II, Academic Press, NY).

[0226] According to a further aspect of the present invention there is provided a peptide ligand as defined herein for use in inhibiting or treating a disease or condition mediated by a bacterial infection, or providing prophylaxis to a subject at risk of infection.

[0227] According to a further aspect of the present invention, there is provided a method of inhibiting or treating a disease or condition mediated by bacterial infection, or providing prophylaxis to a subject at risk of infection, comprising administering to a patient in need thereof a peptide ligand as defined herein.

[0228] The peptide ligands of the present invention or pharmaceutical compositions comprising the peptide ligands can be used to treat skin and soft tissue infections, gastrointestinal infections, urinary tract infections, pneumonia, sepsis, intraperitoneal infections, and obstetric / gynecological infections. The infections may be caused by gram-positive bacteria (such as Streptococcus pneumoniae) or gram-negative bacteria (such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii), and may also be caused by more than one bacterial species.

[0229] In one embodiment, the bacterial infection mediated disease or condition is selected from:

[0230] Whooping cough (probably caused by Bordetella pertussis);

[0231] Tetanus (probably caused by Clostridium tetani);

[0232] Diphtheria (probably caused by Corynebacterium diphtheriae);

[0233] Echinococcus (may be caused by the tapeworm Echinococcus);

[0234] Diarrhea, hemolytic uremic syndrome, or urinary tract infection (possibly caused by E. coli);

[0235] Respiratory tract infection or meningitis (possibly caused by Haemophilus influenzae);

[0236] Gastritis, peptic ulcer disease, or gastric tumors (which may be caused by Helicobacter pylori);

[0237] Tuberculosis (may be caused by Mycobacterium tuberculosis);

[0238] Meningitis, pneumonia, bacteremia, or otitis media (possibly caused by pneumococci);

[0239] Food poisoning (possibly caused by Salmonella);

[0240] Shigellosis or gastroenteritis (which may be caused by Shigella bacteria); and

[0241] Cholera (probably caused by Vibrio cholerae).

[0242] The term "inhibit" as referred to herein refers to administration of a composition after an induction event but prior to clinical manifestation of the disease. "Treatment" involves administration of a protective composition after disease symptoms become evident.

[0243] Animal model systems exist that can be used to screen peptide ligands for effectiveness in preventing or treating disease.

[0244] The present invention is further described below with reference to the following examples.

[0245] Example

[0246] Materials and methods

[0247] Peptide synthesis

[0248] The synthesis of peptides was based on Fmoc chemistry using a Symphony peptide synthesizer produced by Peptide Instruments and a Syro II synthesizer produced by MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) were used with appropriate side chain protecting groups: standard coupling conditions were used in each case, followed by deprotection using standard methods.

[0249] Alternatively, the peptide is purified using HPLC and modified with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma) after isolation. To this end, the linear peptide is diluted to about 35 mL with 50:50 MeCN:H2O, about 500 μL of 100 mM TATA in acetonitrile is added, and the reaction is initiated with 5 mL of 1M NH4HCO3 in H2O. The reaction is allowed to proceed at room temperature for about 30 to 60 minutes and lyophilized once the reaction is complete (judged by MALDI). Upon completion, 1 mL of 1M L-cysteine ​​hydrochloride monohydrate (Sigma) in H2O is added to the reaction at room temperature for about 60 minutes to quench any excess TATA.

[0250] After lyophilization, the modified peptide was purified as above, while using a Gemini C18 column (Phenomenex) instead of Luna C8 and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA modified material were pooled, lyophilized and stored at -20°C.

[0251] Unless otherwise stated, all amino acids were used in the L-configuration.

[0252] In some cases, the peptide was converted to an activated disulfide prior to coupling to the free thiol group of the toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol equiv) was added to a solution of the peptide (20 mM) in dry DMSO (1 mol equiv). The reaction was mixed well and DIPEA (20 mol equiv) was added. The reaction was monitored by LC / MS until completion.

[0253] Biological data

[0254] Fluorescence polarization direct binding assay

[0255] Fluorescence polarization was performed using fluorescein-labeled peptides and unmodified PBP proteins and measured using a BMG Labtech PHERAstar FS equipped with a FP 485 520520 optical module.

[0256] 10 mM fluorescent peptide in DMSO was diluted to 2.5 nM in binding buffer (10 mM HEPES, pH 8, 300 mM NaCl, 2% glycerol). Two-fold serial dilutions of PBP protein were then prepared across 12 wells in binding buffer, with the highest concentration being 21 μM and the lowest concentration being 17 nM.

[0257] Add 10 μl of diluted fluorescent peptide (2.5 nM) to 384-well NBS TM 12 wells of a low volume microplate (Fisher Scientific). 10 μl of serial dilutions of PBP were then added to the wells containing the fluorescent peptide and 5 μl of binding buffer was added to bring the total volume to 25 μl, resulting in a final concentration of 1 nM peptide tracer. Control wells lacking PBP protein were prepared in binding buffer with a final peptide concentration of 1 nM and a final volume of 25 μl. Fluorescence polarization was measured every 5 minutes for 1 hour at room temperature. Gain and focal height were optimized using control wells lacking protein. Wells were excited at 485 nm and emission detection was set to 520 nm.

[0258] Data were analyzed in GraphPad software to derive values ​​for dissociation constants. Experiments were repeated at least three times.

[0259] Certain peptide ligands of the invention (having fluorescein attached to the C-terminus with a Sar6-lysine linker) were tested in the above binding assays and the results for binding to the indicated PBPs are shown in Table 1:

[0260] Table 1: Direct binding data for selected peptide ligands of the invention

[0261] Peptide number Peptide sequence Kd(μM) PBP BCY9377 A-(SEQ ID NO: 1)-A 0.31 1a BCY9378 A-(SEQ ID NO: 2)-A 0.08 1a BCY9381 A-(SEQ ID NO: 3)-A 3.95 1a BCY9382 A-(SEQ ID NO: 4)-A 2.37 1a BCY9383 A-(SEQ ID NO: 5)-A 0.40 1a BCY9384 A-(SEQ ID NO: 6)-A 1.32 1a BCY9385 A-(SEQ ID NO: 7)-A 1.02 1a BCY9386 A-(SEQ ID NO: 8)-A 0.87 1a BCY9387 A-(SEQ ID NO:9)-A 3.14 1a BCY9388 A-(SEQ ID NO: 10)-A 0.57 1a BCY9389 A-(SEQ ID NO: 11)-A 0.79 1a BCY9391 A-(SEQ ID NO: 12)-A 0.45 1a BCY9226 A-(SEQ ID NO: 13)-A 3.75 1b BCY9227 A-(SEQ ID NO: 14)-A 2.15 1b BCY9229 A-(SEQ ID NO: 15)-A 2.24 1b BCY9233 A-(SEQ ID NO: 16)-A 0.37 1b BCY9237 A-(SEQ ID NO: 17)-A 0.36 1b BCY9238 A-(SEQ ID NO: 18)-A 1.35 1b BCY12130 A-(SEQ ID NO: 20)-A 2.00 3 BCY14613 A-(SEQ ID NO: 28)-A 0.131 1b BCY13797 A-(SEQ ID NO: 29)-A 0.095 1b BCY14618 A-(SEQ ID NO: 30)-A 0.142 1b BCY14621 A-(SEQ ID NO: 31)-A 0.239 1b BCY14619 A-(SEQ ID NO: 32)-A 0.049 1b BCY14627 A-(SEQ ID NO: 33)-A 0.158 1b BCY14629 A-(SEQ ID NO: 34)-A 0.059 1b BCY14631 A-(SEQ ID NO: 35)-A 0.062 1b BCY14641 A-(SEQ ID NO: 36)-A 0.163 1b BCY14381 A-(SEQ ID NO: 37)-A 1.386 1b

[0262] The control inhibitor, Bocillin, binds to E. coli PBP3 with a Kd of 0.44 μM. In this assay, BCY12130 was further tested for binding to PBPs from related species (Pseudomonas aeruginosa and Acinetobacter baumannii). No binding of BCY12130 to PBP3 from Pseudomonas aeruginosa or Acinetobacter baumannii was observed, indicating selectivity for E. coli PBP3 (see Figure 1 ). Thus, the data demonstrate that the peptide ligands of the invention selectively bind to PBP with high affinity. It is noteworthy that the direct binding values ​​shown herein utilize peptide ligands conjugated to fluorescent tracer molecules.

[0263] Fluorescence polarization competition binding assay

[0264] Method 1

[0265] Fluorescence polarization competition using BODIPY-labeled penicillin tracer and unlabeled peptides in competition with unmodified PBP protein. Polarization was measured using a BMG Labtech PHERAstar FS equipped with a FP 485 520 520 optical module.

[0266] Dilute 5 mM fluorescent BODIPY-labeled penicillin in DMSO to 6.25 nM in binding buffer (10 mM HEPES, pH 8, 300 mM NaCl, 2% glycerol). Dilute unmodified PBP to 2 μM in binding buffer. Prepare two-fold serial dilutions of unmodified peptide in binding buffer across 12 wells with a final well concentration of 60 μM at the highest and 50 nM at the lowest. Add 5 μl of the serial dilutions of unmodified peptide or carbenicillin to 384-well NBS TM 12 wells of a low-volume microplate (FisherScientific). Then 10 μl of diluted fluorescent BODIPY-labeled penicillin (6.25 nM) was added to the 12 wells containing the unmodified peptide dilution. Then 10 μl of unmodified PBP (2 μM) was added to the 12 wells containing the unmodified peptide and fluorescent BODIPY-labeled penicillin, making the total volume reach 25 μl, the final concentration of fluorescent BODIPY-labeled penicillin reached 2.5 nM, and the unmodified PBP reached 800 nM.

[0267] Prepare control wells lacking unmodified peptide, with a final concentration of 2.5 nM fluorescent BODIPY-labeled penicillin and 800 nM unmodified PBP in binding buffer in a final volume of 25 μl. Prepare a second control well lacking unmodified peptide and unmodified PBP in binding buffer with a final concentration of 2.5 nM fluorescent BODIPY-labeled penicillin and 25 μl in a final volume.

[0268] Fluorescence polarization was measured every 5 minutes for 1 hour at room temperature. Gain and focal height were optimized using control wells lacking unmodified peptide and unmodified PBP. Wells were excited at 485 nm and emission detection was set at 520 nm.

[0269] Data were analyzed in GraphPad software to derive the value of inhibition constant. Experiments were repeated at least three times.

[0270] Certain peptide ligands of the present invention were tested in the above competition assay, and the results are shown in Table 2:

[0271] Table 2: Competition binding data for selected peptide ligands of the invention

[0272] Peptide number Peptide sequence Ki(μM) BCY12130 A-(SEQ ID NO: 19)-A 0.24 BCY12132 A-(SEQ ID NO: 20)-A 0.57

[0273] Figure 2Further data from this assay for BCY12130 is shown in . From the data presented herein, it can be seen that BCY12130 outperforms Bocillin in binding to E. coli PBP3. BCY12130 exhibits a higher Ki (0.24 μM) for Bocillin competition than the control inhibitor carbenicillin (0.52 μM). Thus, the peptide ligands of the present invention selectively bind to and inhibit the β-lactam binding of PBP.

[0274] Method 2

[0275] Fluorescence polarization competition was performed using BCY9378 as a tracer (and with fluorescein attached to the C-terminus with a Sar6-lysine linker) and unlabeled peptides, competing for unmodified PBP protein. Polarization was measured using a BMG Labtech PHERAstar FS equipped with a FP 485 520 520 optical module.

[0276] Dilute BCY9378 (5 mM in DMSO) to 6.25 nM in binding buffer (10 mM HEPES, pH 8, 300 mM NaCl, 2% glycerol). Dilute unmodified PBP protein to 2 μM in binding buffer. Prepare two-fold serial dilutions of unmodified peptide in binding buffer across 12 wells with a final well concentration of 60 μM at the highest and 50 nM at the lowest. Add 5 μl of the serial dilutions of unmodified peptide to the 384-well NBS TM 12 wells of a low-volume microplate (Fisher Scientific) were added. 10 μl of diluted BCY9378 (6.25 nM) was then added to the 12 wells containing the unmodified peptide dilution. 10 μl of unmodified PBP (2 μM) was then added to the 12 wells containing the unmodified peptide and BCY9378, bringing the total volume to 25 μl, the final concentration of BCY9378 to 2.5 nM, and the unmodified PBP to 800 nM.

[0277] Prepare control wells lacking unmodified peptide, with a final BCY9378 concentration of 2.5 nM and unmodified PBP concentration of 800 nM in binding buffer in a final volume of 25 μl. Prepare a second control well lacking unmodified peptide 30 and unmodified PBP in binding buffer with a final BCY9378 concentration of 2.5 nM and a final volume of 25 μl.

[0278] Fluorescence polarization was measured every 5 minutes for 1 hour at room temperature. Gain and focal height were optimized using control wells lacking unmodified peptide and unmodified PBP. Wells were excited at 485 nm and emission detection was set at 520 nm.

[0279] Data were analyzed in GraphPad software to derive the value of inhibition constant. Experiments were repeated at least three times.

[0280] Certain peptide ligands of the present invention were tested in the above competition assay, and the results are shown in Table 3:

[0281] Table 3: Competition binding data for selected peptide ligands of the invention

[0282] Peptide number Peptide sequence Ki(μM) BCY10028 A-(SEQ ID NO: 21)-A 0.61382 BCY10027 A-(SEQ ID NO: 22)-A 0.11 BCY10026 A-(SEQ ID NO: 23)-A 0.55475 BCY10025 A-(SEQ ID NO: 24)-A 0.06668 BCY10024 A-(SEQ ID NO: 25)-A 0.19585 BCY10022 A-(SEQ ID NO: 26)-A 1.1667 BCY10020 A-(SEQ ID NO: 27)-A 0.6019

[0283] Minimum inhibitory concentration and minimum bactericidal concentration determination

[0284] Minimum inhibitory concentration (MIC) assays were performed using an E. coli strain from the Zgurskaya lab that was engineered to have inducible pores to render the outer membrane permeable (Krishnamoorthy et al. (2016) doi: https: / / doi.org / 10.1128 / AAC.01882-16 ). Strains used: GKCW101; GKCW102; GKCW103; and GKCW104.

[0285] First, an overnight culture of bacteria was prepared by transferring a single bacterial colony into 5 mL of cation-regulated Mueller-Hinton broth (CA-MHB) supplemented with 50 μg / mL kanamycin. The next day, the overnight culture was diluted 1 / 100 in 25 mL of CA-MHB with 50 μg / mL kanamycin and cultured until the optical density reached 0.3 as measured at 600 nm on a spectrometer.

[0286] Filter-sterilized arabinose was then added to a concentration of 0.1% w / v, followed by incubation until the optical density at 600 nm was equal to 1.

[0287] The culture medium was then diluted to 1 × 10 6 CFU mL -1 .

[0288] In a 96-well microtiter plate, 100 μL of CA-MHB was dispensed into wells in columns 2-12 and 200 μL was added to wells in column 1 to prepare 2-fold serial dilutions.

[0289] Then add up to 8 μl of peptide ligand to the wells in column 1 and dilute twice on the plate. Positive controls (effective antibiotics) and DMSO controls are included in rows G and H, respectively. The plate is then sealed with a breathable seal and incubated at 37°C for 18 hours. The optical density of each well at 600nm is then measured using a PheraStar FSx plate reader. The MIC value is determined as the cutoff concentration between visible bacterial growth and no growth.

[0290] The minimum bactericidal concentration (MBC) was determined by dispensing 5 μL of the MIC culture from each well onto large LB agar plates (100 mL) followed by incubation overnight at 37° C. The MBC was calculated as the antibiotic concentration at which no colonies were detected on the agar.

[0291] Alternatively, 10 μ l MIC assays are performed using Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 19585) and Enterobacter cloacae (NCTC 13405) to assess the MIC of peptide ligands. The test compound is dissolved in the desired solvent at high concentrations. In the case of using DMSO to dissolve the test compound, a DMSO control with an equivalent DMSO concentration is included for each test organism. For each test organism, a control antibiotic (known test organism susceptibility to control antibiotics) is tested in parallel. This allows comparison of results between runs and validation of test procedures and organisms.

[0292] Using sterile technique, 2-fold serial dilutions (across a range of 10 points) of the test compound were prepared in 5 μl of cation-adjusted Muller Hinton liquid medium (CA-MHB) on the inverted lid of a 96-well plate. An inoculum of each test organism was prepared in phosphate-buffered saline (PBS) to match the turbidity of a 0.5 McFarland standard and then diluted 100-fold. Each well (except the negative control well) was inoculated with 5 μl of inoculum. For each test antibiotic, a negative control consisting of only CA-MHB (no bacteria) was included; and a positive control containing only CA-MHB and bacteria (no antibiotics). The bottom of the 96-well plate was used as a lid, and the plate was incubated in a humidifying box to reduce evaporation. A humidifying box was constructed by sealing the test plate in a preheated plastic box filled with paper towels saturated with PBS. The test plate was incubated at the MIC determination temperature and time period specified in the CLSI guidelines. The droplets were illuminated using white light irradiated through the bottom of the plate, and the MIC was evaluated based on turbidity.

[0293] Certain peptide ligands of the invention were tested in the above inhibitory and bactericidal concentration assays along with the control antibiotic carbenicillin, and the results are shown in Tables 4 and 5:

[0294] Table 4: MIC data of selected peptide ligands of the present invention

[0295]

[0296] Table 4 continued:

[0297]

[0298] “nt” indicates not tested, and values ​​with ">” indicate that no inhibition was observed at the indicated highest concentration of the peptide ligand tested.

[0299] Table 5: MBC data of selected peptide ligands of the present invention

[0300]

[0301] Values ​​with a ">" indicate that no bactericidal effect was observed at the indicated highest concentration of the peptide ligand tested.

[0302] These data demonstrate the ability of the peptide ligands of the invention to inhibit bacterial growth of E. coli and the closely related species Salmonella typhimurium and Enterobacter cloacae. Although the peptide ligand BCY12130 had no bactericidal effect on the E. coli strain GKCW103, which lacks an efflux pump, a bacteriostatic effect was observed.

[0303] Cytotoxicity assay

[0304] The peptides were tested in a cytotoxicity assay to determine the toxicity of the peptides to human cell lines. Luminescence values ​​were recorded as a measure of cell viability and analyzed using Ultra-Glo TM Luciferase catalyzes the ATP-dependent conversion of luciferin to oxyluciferin for assay.

[0305] Cell lines were maintained in appropriate culture media according to ATCC guidelines for specific cell types (in this case HepG2#HB-8065 and HT-1080#CCL-121). Cells were seeded at a density of 10,000 cells per well in a 96-well opaque-sided plate (VWR734-1660) 24 hours prior to the addition of peptides. Plates were incubated overnight at 37°C, 5% CO2. The next day, peptides were diluted to a final DMSO concentration of 0.5% (final peptide concentration of 40-80 μM) in the appropriate cell line culture media. Culture media was removed from wells containing cells and replaced with culture media containing peptides. Control wells were included containing: cells only, cells + 0.5% DMSO, or cells + 11 μM staurosporine (Sigma #S5921, a known protein kinase inhibitor). Treated plates were incubated at 37°C, 5% CO2 for 72 hours. After this time, 100 μl CellTitre- (Promega #G7570) was added to the wells and incubated with shaking for 10 minutes to induce lysis. Luminescence values ​​were read using a BMG Labtech PHERAstar FS equipped with a LUMplus optical module.

[0306] Data were analyzed in GraphPad software to determine viable cells expressed as the percentage of wells containing cells + 0.5% DMSO. Values ​​quoted are representative of two independent experiments.

[0307] Certain peptide ligands of the invention (BCY12130 and BCY12132) were tested in the above described cytotoxicity assay and no cytotoxicity towards human cell lines was observed at the highest concentrations tested (54 μM and 56 μM peptide ligands, respectively).

[0308] Morphological test

[0309] Bacterial growth in a volume of 2 μl was placed on a slide (Hendley multispot microscope slide) in the absence or presence of unmodified peptide ligand and heat fixed at 45°C. The slides were Gram stained by adding crystal violet for 30 seconds, Gram iodine for 30 seconds, and acetone for 2 seconds. Counterstaining was performed with safranin for 30 seconds. Between each stage, the slides were thoroughly washed with water. Images were observed using a Zeiss Axiocam ERc 5s under oil immersion (×100 magnification).

[0310] Each droplet was compared to a growth control without antimicrobial agent to detect differences in cell morphology.

[0311] Certain peptide ligands of the invention were tested in the above-mentioned morphological assays and the results are shown in Figure 3 middle.

[0312] These data show that incubation of E. coli and the closely related species Salmonella typhimurium and Enterobacter cloacae in the presence of sub-MIC concentrations (13.75 μg / ml and 27.5 μg / ml) of BCY12130 resulted in filamentous growth, indicating that BCY12130 is a PBP3 inhibitor.

[0313] In summary, the data presented herein demonstrate that the peptide ligands of the present invention bind to PBP3 of E. coli, inhibit β-lactam binding, and inhibit bacterial growth of E. coli and closely related strains by targeting cell division. Sequence Listing <110> Baisco Technology Development Co., Ltd. <120> Bicyclic peptide ligands that bind to PBP <130> BIC-C-P2622PCT <150> GB1912320.7 <151> 2019-08-28 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Cys Arg Phe Ser Ser Cys Pro Pro Tyr His Val Cys 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Cys Pro Tyr Thr Ser Cys Pro Pro His Thr Met Cys 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 3 Cys His Pro Arg His Gln Glu Gly Tyr Cys Met Pro Cys 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 4 Cys Tyr Asn His Lys Trp Gly Ala Met Cys Thr His Pro Cys 1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Cys His Asp Trp Asp Tyr Arg His Leu Cys Tyr Trp Arg Cys 1 5 10 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Cys Asp Ile Tyr Arg Glu Cys His Tyr Thr Ser Trp Ser Val Cys 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 Cys Lys Pro Ser Leu Ser Cys Gln His Leu Pro Arg Ala Leu Cys 1 5 10 15 <210> 8 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 8 Cys Pro Phe Thr Gly Pro Cys Arg Pro His Tyr Ile Cys 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 9 Cys Tyr Thr Ser Cys Pro Glu His His Val Phe Ala Cys 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 10 Cys Asp Asn Cys Trp Glu Arg Gln Trp Tyr Ala Cys 1 5 10 <210> 11 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Cys Asn Pro Arg Cys His Pro Val Tyr Thr Ser Phe Phe Cys 1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 Cys Gly Ala Pro Cys Arg Pro His Tyr Val Pro Trp Phe Cys 1 5 10 <210> 13 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 Cys Val Tyr Ala Pro Glu Asn Leu Leu Cys Gly Ser Cys 1 5 10 <210> 14 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 14 Cys Ser Asn Pro Thr Cys Val Tyr Thr Pro Thr Asn Leu Phe Cys 1 5 10 15 <210> 15 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 15 Cys Asn Thr Cys Ile Tyr Ala Ser Glu Asn Leu Leu Cys 1 5 10 <210> 16 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 16 Cys Ser Ala Thr Trp Gly Ser Arg Ser Cys Pro Val Lys Phe Cys 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Cys Pro Asn Ala Cys Trp Thr Val His Tyr Ser Gly Tyr Gln Cys 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Cys His Glu Phe Ser Leu Asp Cys Ile Leu Phe Gly Thr Ser Cys 1 5 10 15 <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Cys Ser Phe Pro Lys Cys Pro Trp Val Glu Gly Cys 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Cys Arg Thr Phe Gly Cys Trp Trp Glu Gly Cys 1 5 10 <210> twenty one <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one Cys Pro Pro Val Cys Arg Pro His Tyr Val His Trp Met Cys 1 5 10 <210> twenty two <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty two Cys Pro Val Gly Cys Arg Pro His Tyr Val His Trp Ser Cys 1 5 10 <210> twenty three <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty three Cys Arg Tyr Thr Ser Cys Pro Pro Tyr Thr Val Cys 1 5 10 <210> twenty four <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty four Cys Pro Tyr Thr Ser Cys Pro Pro Tyr Thr His Cys 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Cys Pro Tyr Thr Thr Cys Pro Pro Tyr His Ala Cys 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Cys Val Phe Thr Thr Cys Pro Pro Tyr Thr Val Cys 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Cys Thr Tyr Thr Thr Cys Pro Pro Phe Thr Ile Cys 1 5 10 <210> 28 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Cys Trp Gly Ser Trp Arg Cys Pro Ile Val His Ser Cys 1 5 10 <210> 29 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 29 Cys Trp Gly Ser Leu Arg Cys Pro Ile Val His Ser Cys 1 5 10 <210> 30 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 30 Cys Trp Gly Ser Leu Arg Cys Pro Ile His Tyr Ser Cys 1 5 10 <210> 31 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Cys Trp Gly Ser Leu Arg Cys Pro Ile Lys Trp Asp Cys 1 5 10 <210> 32 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 32 Cys Trp Gly Ser Leu Arg Cys Pro Ile Thr Ala His Cys 1 5 10 <210> 33 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 33 Cys Trp Gly Ser Lys Ala Cys Pro Ile Thr Trp His Cys 1 5 10 <210> 34 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 34 Cys Trp Gly Ser Arg Gln Cys Pro Ile Ser Trp Thr Cys 1 5 10 <210> 35 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 35 Cys Trp Gly Thr Gln Lys Cys Pro Val Gly Tyr Trp Cys 1 5 10 <210> 36 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 36 Cys Trp Gly Ser Lys Ser Cys Pro Ile Thr Trp Lys Cys 1 5 10 <210> 37 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 37 Cys Trp Gly Thr Ser Ala Cys Pro Val Thr His Glu Cys 1 5 10

Claims

1. A peptide ligand capable of binding to a penicillin binding protein (PBP) or a pharmaceutically acceptable salt thereof, wherein the peptide ligand comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two loop sequences, and the molecular scaffold forms a covalent bond with the cysteine ​​residues of the polypeptide, so that at least two polypeptide loops are formed on the molecular scaffold, wherein (a) the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA); and (b) the PBP is (i) Streptococcus pneumoniae PBP1a, and the polypeptide has an amino acid sequence selected from the group consisting of: C i RFSSC ii PPYHVC iii (SEQ ID NO:1); C i PYTSC ii PPHTMC iii (SEQ ID NO:2); C i HPRHQEGYC ii MPC iii (SEQ ID NO:3); C i YNHKWGAMC ii THPC iii (SEQ ID NO:4); C i HDWDYRHLC ii YWRC iii (SEQ ID NO:5); C i DYREC ii HYTSWSVC iii (SEQ ID NO:6)? C i KPSLSC ii QHLPRALC iii (SEQ ID NO:7); C i PFTGPC ii RPHYIC iii (SEQ ID NO:8); C i YTSC ii PEHHVFAC iii (SEQ ID NO:9); C i DNC ii WERQWYAC iii (SEQ ID NO:10); C i NPRC ii HPVYTSFFC iii (SEQ ID NO:11); C i GAPC ii RPHYVPWFC iii (SEQ ID NO:12); C i PVC ii RPHYVHWMC iii (SEQ ID NO:21); C i PVGC ii RPHYVHWSC iii (SEQ ID NO:22); C i RYTSC ii PPYTVC iii (SEQ ID NO:23); C i PYTSC ii PPYTHC iii (SEQ ID NO:24); C i PYTTC ii PPYHAC iii (SEQ ID NO:25); C i VFTTC ii PPYTVC iii (SEQ ID NO:26); and C i TYTTC ii PPFTIC iii (SEQ ID NO:27); or (ii) Escherichia coli PBP1b, and the polypeptide has an amino acid sequence selected from the group consisting of: C i VYAPENLLC ii GSC iii (SEQ ID NO:13); C i SNPTC ii VYTPTNLFC iii (SEQ ID NO:14); C i NTC ii IYASENLLC iii (SEQ ID NO:15) C i SATWGSRSC ii PVKFC iii (SEQ ID NO:16): C i PNAC ii WTVHYSGYQC iii (SEQ ID NO:17); C i HEFSLDC ii ILFGTSC iii (SEQ ID NO:18); C i WGSWRC ii PIVHSC iii (SEQ ID NO:28); C i WGSLRC ii PIVHSC iii (SEQ ID NO:29); C i WGSLRC ii PIHYSC iii (SEQ ID NO:30); C i WGSLRC ii PIKWDC iii (SEQ ID NO:31); C i WGSLRC ii PITAHC iii (SEQ ID NO:32); C i WGSKAC ii PITWHC iii (SEQ ID NO:33); C i WGSRQC ii PISWTC iii (SEQ ID NO:34); C i WGTQKC ii PVGYWC iii (SEQ ID NO:35); C i WGSKSC ii PITWKC iii (SEQ ID NO:36); and C i WGTSAC ii PVTHEC iii (SEQ ID NO:37); or (iii) Escherichia coli PBP3, and the polypeptide has an amino acid sequence selected from the group consisting of: C i SFPKC ii PWV iii (SEQ ID NO: 19); and C i RTFGC ii WWEGC iii (SEQ ID NO:20), Among them C i , C ii and C iii The first, second and third cysteine ​​residues are indicated.

2. The peptide ligand as defined in claim 1 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium, potassium, calcium and ammonium salts.

3. The peptide ligand as defined in claim 1 or a pharmaceutically acceptable salt thereof, wherein the PBP is Escherichia coli PBP3, and the polypeptide has an amino acid sequence C i SFPKC ii PWV iii (SEQ ID NO: 19).

4. A peptide ligand as defined in claim 3 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium, potassium, calcium and ammonium salts.

5. A pharmaceutical composition comprising the peptide ligand according to claim 1 or claim 2 or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable excipients.

6. A pharmaceutical composition as defined in claim 5, further comprising one or more therapeutic agents.

7. A pharmaceutical composition comprising the peptide ligand according to claim 3 or claim 4 or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable excipients.

8. A pharmaceutical composition as defined in claim 7, further comprising one or more therapeutic agents.

9. Use of a peptide ligand as defined in claim 3 or claim 4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 7 or claim 8, in the preparation of a medicament for inhibiting or treating a disease or condition mediated by an Escherichia coli bacterial infection, or for providing prevention to a subject at risk of such infection.

Images