WO2021195716A1 - Antibiotic conjugates - Google Patents

Antibiotic conjugates Download PDF

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Publication number
WO2021195716A1
WO2021195716A1 PCT/AU2021/050306 AU2021050306W WO2021195716A1 WO 2021195716 A1 WO2021195716 A1 WO 2021195716A1 AU 2021050306 W AU2021050306 W AU 2021050306W WO 2021195716 A1 WO2021195716 A1 WO 2021195716A1
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Prior art keywords
vancomycin
conjugate
pharmaceutically acceptable
amino acid
peptide
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PCT/AU2021/050306
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French (fr)
Inventor
Max CRYLE
Jennifer PAYNE
Julien Tailhades
Anton PELEG
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Monash University
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Priority claimed from AU2020901025A external-priority patent/AU2020901025A0/en
Application filed by Monash University filed Critical Monash University
Publication of WO2021195716A1 publication Critical patent/WO2021195716A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
    • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to conjugates comprising a glycopeptide antibiotic agent and a chemotactic formylated peptide and their use in the treatment or prevention of bacterial infections.
  • the conjugates of the invention are particularly suited to the prevention or treatment of bacterial infections caused by Staphylococcus aureus.
  • Antibiotic resistance is an increasing threat worldwide with widespread antibiotic use.
  • Some strains such as methicillin-resistant Staphylococcus aureus (MRSA) have developed multidrug resistance (MDR), raising significant concern in the medical community.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MDR multidrug resistance
  • S. aureus is a well-adapted human pathogen, which expresses many virulence factors that allow it to colonise tissues and evade and resist the immune system. With added antibiotic resistance S. aureus represents a deadly threat to hospital patients, who are immunocompromised or present open wounds.
  • Vancomycin is often used as the last resort treatment for MRSA (Liu, C.; et al., Clin. Infect. Dis. 2011, 52 (3), el8-e55), as these bacteria are resistant to b-lactam antibiotics.
  • Successful control of antibiotic resistant bacteria is dependent on a variety of strategies, including identifying new targets, deepening understanding of underlying resistance mechanisms, optimising existing drugs, rational drug design and combinatorial biology (Hughes, D. Nat. Rev. Genet. 2003, 4 (6), 432-41).
  • the body’s innate immune response against bacterial pathogens like S. aureus is the primary route for the clearance of such infections.
  • Neutrophils are the major effectors of the innate immune response against infection and comprise approximately 50-70% of all leukocytes in the bloodstream (Mocsai, A., J. Exp. Med. 2013, 210 (7), 1283). Individuals with low neutrophil count (neutropenia) are more susceptible to infection as a consequence (Gibson, C.; Hopkins, N., Blood 2014, 124 (8), 1251-1258).
  • Neutrophils are well-equipped to kill bacterial pathogens directly and have three main ways of doing so: phagocytosis, degranulation and neutrophil extracellular traps (NETs).
  • ROS reactive oxygen species
  • H2O2 superoxide anion
  • HOC1 hypochlorous acid
  • neutrophils release granules containing an array of proteolytic enzymes and membrane -permeabilising proteins and peptides, including elastase, lysozyme, defensin and bactericidal/permeability-increasing protein (BPI) (Cowland, J. B.; Borregaard, N., Immunol. Rev. 2016, 273 (1), 11-28).
  • BPI bactericidal/permeability-increasing protein
  • These granules also contain NADPH oxidase and myeloperoxidase (MPO), which assist in generating ROS.
  • Neutrophils are also capable of killing and inhibiting the growth of bacteria by forming NETs.
  • NETs are comprised of antimicrobial peptides and enzymes bound to a mesh of DNA, and are formed when neutrophils are stimulated by a range of factors including bacterial pathogens and ROS (Arazna, M.; Pruchniak, M. P.; Demkow, U., Respir. Physiol. Neurobiol. 2013, 187 (1), 74-77).
  • This additional anti-bacterial mechanism can immobilise and kill bacteria that cannot be phagocytosed.
  • Neutrophils patrol the body through the bloodstream, and are recruited from circulation to an infected site by chemical signals.
  • Chemokines are signalling proteins secreted by cells that induce chemotaxis, i.e. movement in the direction of an increasing chemical gradient. Endogenous signals released by the host as well as some foreign signals induce chemotaxis in neutrophils.
  • GPCRs G-protein coupled receptors
  • LTB4 leukotriene B4
  • S. aureus can also inhibit the key neutrophil functions of killing by ROS, phagocytosis and chemotaxis.
  • S. aureus can reduce the damage of ROS by expression of superoxide dismutase and staphyloxanthin, the yellow carotenoid produced by S. aureus responsible for its golden colour (Liu, G. Y.; et al., J. Exp. Med. 2005, 202 (2), 209-215).
  • Staphyloxanthin acts as an antioxidant, neutralising the ROS produced in the phagosome of the neutrophil.
  • the phagocytosis of bacteria by neutrophils requires recognition of pathogen-associated molecular patterns (PAMPs) on the surface of the bacteria by pattern recognition receptors (PRRs). Phagocytosis is greatly enhanced when opsonins such as IgG antibodies and complement components are bound to the bacteria.
  • PAMPs pathogen-associated molecular patterns
  • PRRs pattern recognition receptors
  • the polysaccharide capsule surrounding most S. aureus strains can prevent the binding of opsonins (Foster, T. J., Nat. Rev. Microbiol. 2005, 3 (12), 948-958).
  • a surface protein on S. aureus, protein A can bind antibodies via their Fc domain (the region that is recognised by neutrophils), which leads to reduced phagocytosis.
  • S. aureus can express other cell wall- anchored proteins that promote adhesion to the extracellular matrix and form biofilms. These include clumping factor A (clfA), collagen adhesin (Cna) and biofilm-associated protein (Bap) (Foster, T. J.; et al., Nat. Rev. Microbiol. 2014, 12 (1), 49-62). These virulence factors offer protection from phagocytosis, thereby inhibiting the ability of neutrophils to recognise and kill S. aureus.
  • chemotaxis inhibitor protein of S. aureus a virulence factor known as chemotaxis inhibitor protein of S. aureus (CHIPS), that binds to FPR and C5a receptor, and antagonise their ability to induce chemotaxis (Postma, B.; et al., J. Immunol. 2004, 172 (11), 6994-7001).
  • FLIPr formyl peptide receptor-like 1 inhibitory protein
  • the present invention provides a conjugate of formula (I): or a pharmaceutically acceptable salt thereof, wherein GPA is a glycopeptide antibiotic agent;
  • L is a linker moiety
  • FP is a chemotactic formylated peptide.
  • the invention provides a formylated peptide, or pharmaceutically acceptable salt thereof, selected from:
  • R 1 represents a side chain of an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline;
  • R 2 represents a side chain of an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine;
  • R 3 represents a side chain of an amino acid selected from tyrosine, aspartic acid, 4- fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
  • R 4 represents a side chain of an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
  • R 4a represents a side chain of an amino acid selected from leucine, arginine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
  • R 5 is selected from NH 2 , OH, or SH.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a conjugate of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
  • the invention provides a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate of the invention, or a pharmaceutically acceptable salt thereof.
  • a further aspect the invention provides a conjugate of the invention, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a bacterial infection in a subject in need thereof.
  • Another aspect the invention provides use of a conjugate according to the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a bacterial infection in a subject in need thereof.
  • Figure 1 Antibacterial activity of formylated peptide-vancomycin conjugates.
  • FIG. 2 Chemotactic activity of the conjugates of formylated peptide (fMLFG, FP1) linked to vancomycin at the three sites; vancosamine primary amine (V-linked), methylated amine (N-linked); or the carboxyl group (C-linked); with 3 different length PEG linkers of 0 (La), 3 (Lb), or 6 (Lc) repeats.
  • FIG 3 Comparison of binding of vancomycin and conjugate binding to 3 strains of S. aureus. Vancomycin or the formylated peptide conjugated to vancomycin through the C terminus with a 4 repeat PEG linker (FPl-Ld-C-Van) were fluorescently labelled with BODIPY and their binding to the S. aureus was imaged by STED microscopy. Scale bar is l ⁇ m.
  • Figure 4 AiryScan imaging of S.
  • Figure 5 The sequence of the formylated peptide effects neutrophil recruitment.
  • A Schematic of the process of creating and testing a library of formylated peptides based on the fMLFG (FP1) sequence.
  • the library of formylated peptides were grouped into 6 profiles (line graphs, B-F), based on the concentration of the formylated peptide that resulted in the greatest recruitment of neutrophils in a transwell assay. This was a peak observed at: 100 nM (B), at both 10 and 100 nM (C), at 10 nM (D), at 100 and 1000 nM (E), at 1000 nM (F).
  • Chemotaxis was calculated relative to the no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor, n>3, error bars are SEM. Dotted line on graphs represents the media only control recruitment level.
  • Figure 6 The formylated peptide sequence conjugated to vancomycin effects neutrophil recruitment Representative formylated peptides from each of the 6 formylated peptide library profiles were linked to the C-terminus of vancomycin and retested for the ability to recruit neutrophils using the same transwell method (solid bars represent conjugate). This was a peak observed at: 100 nM (A), at both 10 and 100 nM (B), at 10 nM (C), at 100 and 1000 nM (D), at 1000 nM (E). Chemotaxis was calculated relative to the no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor, n>3, error bars are SEM. Dotted line on graphs represents the media only control recruitment level.
  • Figure 7 Peptides that resulted in no recruitment of human neutrophils in a transwell assay at 1, 10, 100, and 1000 nM. Chemotaxis was calculated relative to the no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor, n>3, error bars are SEM. Dotted line is the media only control recruitment level.
  • Figure 8 In silico comparison of LogP (A) and solvent accessible surface area I SASA (B) of formylated peptide FP1 compared to FP2 to FP24.
  • Figure 9 Infection on a chip microfluidics used to monitor neutrophil migration and phagocytosis.
  • A The infection-on-a-chip microfluidic device used to monitor neutrophil migration to compound and phagocytosis of S. aureus over time present in a 6 well plate.
  • B Time course of neutrophils migrating into the microchamber containing S. aureus bioparticles labelled with pHrodo, in the presence of free formylated peptide (FP1 or FP9) or conjugated (FPl-La-C-Van or FP9-La-C-Van) at 1000 nM. Images are representative of four donors, scale bar is 10 pm.
  • Figure 10 Finking a formylated peptide to vancomycin enhances phagocytosis activity of neutrophils.
  • the infection-on-a-chip microfluidic device was used to monitor neutrophil migration and phagocytosis over time.
  • (A) The percentage of neutrophils recruited into the microchamber in the presence of conjugated (triangles, FPl-La-C-Van; FP9-La-C-Van) or free formylated peptide (circles, FP1; FP9, 1000 nM) increased over time. Data is the average of four donors, with error bars of SEM.
  • (B) Area of pHrodo fluorescence increased over time when conjugated (triangles, FPl-La-C-Van; FP9-La-C-Van) or free formylated peptide is present (circles, FP1; FP9, 1000 nM). Data is the average of experiments conducted with four different donors, with error bars of SEM.
  • FIG. 11 Graphical representation of competition binding to human neutrophils between the FPR2 antagonist RhB-PBlO and different formylated peptides and conjugates.
  • the formylated peptide fMLF binds preferentially to FPR1 and was used as a negative control, while the formylated peptides fMVIL which binds preferentially to FPR2 was used as a positive control.
  • the peptides fMLFG (FP1) and fMChaFG (FP9) and their corresponding conjugates were examined for their ability to compete off RhB-PBlO binding to neutrophils.
  • Figure 12 Graphical representation of recruitment of human neutrophils to the teicoplanin aglycone conjugates.
  • a Transwell assay was used to measure the human neutrophil recruitment to the fMLFK (fPep) formylated peptide linked to either the N terminus of teicoplanin aglycon (Teio-N-fPep) or c terminus (Teico-C-fPep). Error bars are SD, results from one donor.
  • Figure 13 The conjugated formylated peptide to vancomycin reduces bacterial load and a mouse pneumonia model.
  • A Eight-week-old female mice infected by intranasal inhalation of 10 7 cfu S. aureus to induce pneumonia. One -hour post infection (hpi) mice were given intranasal therapy at 0.2 mg/mouse equivalent of vancomycin (van, vancomycin; FP1; or FP9-La-C-Van) or vehicle control (control). The lungs where collected 12 hpi.
  • FIG. 14 Conjugate treatment reduces inflammation and alveoli structure is retained in mouse pneumonia model.
  • Eight-week-old female mice were infected by intranasal inhalation of 10 7 cfu S. aureus to induce pneumonia.
  • One -hour post infection (hpi) mice were given intranasal therapy at 0.2 mg/mouse equivalent of vancomycin (Van) or vehicle control (control).
  • the lungs where collected 12 hpi and lung histology performed with haemotoxylin and eosin staining for one mouse per treatment.
  • the formylated peptide FP1 or the conjugate FP9-La-C-Van displayed reduced infiltration of innate immune cells into the alveoli.
  • Black arrows indicate neutrophils
  • white arrows proteinaceous fluid
  • grey arrows indicate cocci bacteria.
  • FIG. 15 Conjugate treatment reduces immune cell infiltrating and alveoli structure is retained in mouse pneumonia model.
  • Eight-week-old female mice were infected by intranasal inhalation of 10 7 cfu S. aureus to induce pneumonia.
  • One-hour post infection (hpi) mice were given intranasal therapy at 0.2 mg/mouse equivalent of vancomycin (Van) or vehicle control (control).
  • the lungs where collected 12 hpi and lung histology performed with haematoxylin and eosin staining for one mouse per treatment. 6 fields of view (FOV) from the outer lobes of the lungs were analysed for the alveoli area (A) or the number of nuclei (B) present.
  • FOV fields of view
  • the conjugate FP9-La-C-Van displayed similar infiltration of innate immune cells into the alveoli and the area of the alveoli as the uninfected normal lung (dotted line). Data is the average of 6 FOV from one mouse lung, with error bars of SEM.
  • Figure 17 Graphical representation of the percentage of neutrophils recruited in the presence of different heat inactivated bacteria alone (control), or with either FP1 or FPl-La-C-Van. The compounds were incubated with the bacteria for 20min and then either washed 3 times (washed, concentration of compound estimated to be less than 10 nM) or loaded directly. Error bars are SEM with data from one biological donor.
  • the present invention provides a conjugate of formula (I): or a pharmaceutically acceptable salt thereof, wherein
  • GPA is a glycopeptide antibiotic agent
  • L is a linker moiety
  • FP is a chemotactic formylated peptide.
  • antibiotic agent will be understood to mean antibiotic agents with structures containing either a glycosylated cyclic or polycyclic nonribosomal peptide, These antibiotics inhibit the cell wall structure of susceptible organisms (principally Gram- positive cocci) by inhibiting peptidoglycan synthesis.
  • First -generation glycopeptide antibiotics include vancomycin, teicoplanin, and ramoplanin; second-generation semi- synthetic glycopeptide antibiotics include oritavancin, dalbavancin, and teiavancin,
  • the term “glycopeptide antibiotic agent” also includes aglycone derivatives of antibiotic agents, for example, vancomycin aglycone or teicoplanin aglycone.
  • the glycopeptide antibiotic agent is selected from vancomycin, vancomycin aglycon, vancomycin desvancosamine, desmethyl vancomycin, dalbavancin, oritavancin, teicoplanin, teiavancin, ramoplanin, decaplanin, chloroeremomycin, teicoplanin A2-2, ristocetin A, eremomycin, balhimycin, actinoidin A, complestanin, chloropeptin 1, kistamycin A, avoparcin, A40926, oritavancin and derivatives thereof.
  • the glycopeptide antibiotic agent is vancomycin.
  • linker as herein used relates to the part of the conjugate that links the glycopeptide antibiotic agent to the chemotactic formylated peptide. It will be understood that the linker should be selected such that it does not compete with the glycopeptide antibiotic agent or the chemotactic formylated peptide.
  • the linker group should be of a length of between 1 nm to 50 nm in order to allow the glycopeptide antibiotic agent to interact with the bacteria unhindered by the chemotactic formylated peptide. In one embodiment, the linker group will comprise one or more polyethelene glycol units.
  • the linker, or subunits of the linker may be amino acid residues, derivatised or functionalised amino acid residues, polyethers, ureas, carbamates, sulphonamides or other subunits that provide adequate distance between the glycopeptide antibiotic agent and the chemotactic formylated peptide without interfering in the function of either group.
  • linker is represented by the formula (II): wherein
  • X is the attachment group between the linker and the glycopeptide antibiotic agent and is selected from -Ci-CioalkylC(O)-, -C 2 -CioalkenylC(0)-, -C 2 -CioalkynylC(0)-, -Ci- CioalkylNH-, -C 2 -CioalkenylNH- -C 2 -CioalkynylNH- -Ci-CioalkylO-, -C 2 -
  • X is an optionally C-terminal amidated amino acid wherein the amino acid is attached to the glycopeptide antibiotic agent via a side-chain functional group; m is 0, 1 or 2; n and p are independently at each occurrence 1 or 2; and denotes the point where the linker is conjugated to the formylated peptide.
  • linker is represented by the formula (III): wherein
  • Y is the point of attachment between the linker and the glycopeptide antibiotic agent and is selected from -C(O)-, -NH-, -0-, or -S- m is 0, 1 or 2; n and p are independently at each occurrence 1 or 2; r is from 1 to 10; and denotes the point where the linker is conjugated to the formylated peptide.
  • linker is represented by a moiety of the formula (XX):
  • each occurrence of R 11 is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue; m is an integer from 1 to 80; and n is an integer from 0 to 1.
  • the linker is represented by a moiety of the formula (XXI): wherein m is an integer from 0 to 40; n is an integer from 0 to 1 ; each occurrence of o is independently an integer from 1 to 5; each occurrence of R 11 is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue.
  • the linker is represented by a moiety of the formula (XXII): wherein m is an integer from 0 to 40; n is an integer from 0 to 1 ; each occurrence of o is independently an integer from 1 to 5; each R 12 is independently NH or O; and each occurrence of R 11 is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue.
  • the conjugates of the invention comprise a chemotactic formylated peptide conjugated to the glycopeptide antibiotic agent via the linker moiety.
  • Formylated peptides are one of the key chemoattractants recognised by neutrophils alongside complement component 5a (C5a) and chemokines.
  • Formylated peptides are found solely in prokaryotic systems including mitochondria, as addition of a formyl group on the N-terminal methionine is a modification present exclusively in prokaryotic proteins.
  • Formylated peptides are released as bacterial waste products or from damaged mitochondria, and are a signal of infection recognised in humans, i.e. pathogen-associated molecular pattern (PAMP).
  • PAMP pathogen-associated molecular pattern
  • N-formyl- methionyl-leucyl-phenylalanine (fMLF), a formylated peptide isolated from the supernatant of Escherichia coli culture, was found to be the most potent stimulator of chemotaxis out of the tripeptides and is often used in standard transwell cell migration assays as a reference against which the chemotactic properties of other substances are tested (Marasco, W. A.; et al., J. Biol. Chem. 1984, 259 (9), 5430-5439).
  • fMIFL N-formyl- methionyl-isoleucyl-phenylalanyl-leucine
  • aureus induces chemotaxis with even greater potency than fMLF (Rot, A.; et al., Proc. Natl. Acad. Sci. U. S. A. 1987, 84 (22), 7967-7971).
  • Formylated peptides are detected by formyl peptide receptor 1 (FPR1) and formyl peptide receptor 2 (FPR2), GPCRs present in neutrophils as well as some other cell types in humans (Fi, F.; et al., J. Leukoc. Biol. 2016, 99 (3), 425-435).
  • FPR1 is the most important receptor of the FPR family for chemotaxis, as it binds the majority of formyl peptides with high affinity and induces chemotaxis with high potency (Bloes, D. A.; Kretschmer, D.; Peschel, A., Nat. Rev. Microbiol. 2015, 13 (2), 95-104).
  • FPR1 shares 69% homology in amino acid sequence with FPR2, but FPR2 binds formyl peptides with much lower affinity (Dorward, D. A.; et al., Am. J. Pathol. 2015, 185 (5), 1172-1184).
  • FPR2 has more functions and can bind a wider variety of ligands while the function of a third member of the FPR family, formyl peptide receptor 3 (FPR3), remains largely unknown.
  • FPR3 formyl peptide receptor 3
  • formylated peptides also stimulate superoxide formation and degranulation from neutrophils as classic antibacterial defences, and hence highlight the importance of formylated peptides in activating host defence mechanisms (Fi, F.; et al., J. Leukoc. Biol. 2016, 99 (3), 425-435).
  • Formylated peptides are the most potent chemoattractants identified to date.
  • the importance of formylated peptides in triggering the immune response has been established in several studies.
  • FPRs are not crucial for healthy host function except when fighting an infection, as a deletion study showed that Fprl knockout mice have a normal healthy phenotype (Gao, J. L.; Lee, E. J.; Murphy, P. M., J. Exp. Med. 1999, 189 (4), 657-662); however, they have significantly delayed neutrophil migration and reduced superoxide production in response to Listeria infection, leading to increased mortality rates.
  • Formylated peptides are necessary for neutrophils to mount a successful immune response as host-derived chemoattractants alone are insufficient. This was also discovered when S. aureus culture supernatant containing deformylated peptides was injected into mice triggered reduced neutrophil migration to the site of injection than the wild type culture supernatant (Di rr, M. C.; et al. Cell. Microbiol. 2006, 8 (2), 207-217). Hence formylated peptides are chemoattractants that play a major role in triggering neutrophil migration.
  • chemotactic formylated peptide refers to a formylated peptide that acts as a chemoattractant that is recognised and results in the chemotaxis of neutrophils, i.e., the neutrophils undergo directed movement along an increasing chemical gradient.
  • the peptides themselves do not undergo movement along an increasing chemical gradient.
  • the formylated peptides act by binding the formyl peptide receptors of neutrophils.
  • the formylated peptide has the sequence: f-MLFG-R 5 - wherein f represents a formyl moiety;
  • R 5 is selected from NH, O or S; and one or two of the residues methionine, leucine, phenylalanine or glycine may be substituted with a naturally or non-naturally occurring amino acid. In another embodiment, one or two of the residues methionine, leucine, phenylalanine or glycine may be substituted with a naturally or non-naturally occurring amino acid as follows:
  • Methionine may be substituted with an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline;
  • Leucine may be substituted with an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine;
  • Phenylalanine may be substituted with an amino acid selected from tyrosine, aspartic acid, 4-fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
  • Glycine may be substituted with an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, proline, or phenylalanine.
  • one of methionine, leucine, phenylalanine or glycine is substituted with naturally or non-naturally occurring amino acid.
  • the formylated peptide, or pharmaceutically acceptable salt thereof has the sequence fMILF-R 5 -, fMIVIL-R 5 - or fMLP-R 5 -; wherein R 5 is as defined above.
  • the formylated peptide, or pharmaceutically acceptable salt thereof is selected from:
  • R 1 represents a side chain of an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline
  • R 2 represents a side chain of an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine
  • R 3 represents a side chain of an amino acid selected from tyrosine, aspartic acid, 4- fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
  • R 4 represents a side chain of an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
  • R 4a represents a side chain of an amino acid selected from leucine, arginine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
  • R 5 is selected from NH 2 , OH, or SH.
  • the formylated peptides defined above are conjugated to the linker via the C-terminus or the derivatised C-terminus defined by R 5 .
  • the formylated peptide, or pharmaceutically acceptable salt thereof is selected from those listed in Table 1:
  • alkyl used either alone or in compound words, denotes straight chain or branched alkyl. Prefixes such as “C2-C10” are used to denote the number of carbon atoms within the alkyl group (from 2 to 10 in this case).
  • straight chain and branched alkyl examples include methyl, ethyl, n -propyl, isopropyl, n-butyl, sec-butyl, /-butyl, n-pcntyl, hexyl, heptyl, 5-methylheptyl, 5-methylhexyl, octyl, nonyl, decyl, undecyl, dodecyl and docosyl (C22).
  • alkenyl used either alone or in compound words, denotes straight chain or branched hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or polyunsaturated alkyl groups as previously defined. Prefixes such as “C2-C20” are used to denote the number of carbon atoms within the alkenyl group (from 2 to 20 in this case).
  • alkenyl examples include vinyl, allyl, 1 -methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 1-hexenyl, 3-hexenyl, 1-heptenyl, 3- heptenyl, 1-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3- butadienyl, 1,4-pentadienyl, 1,3-hexadienyl, 1,4-hexadienyl and 5-docosenyl (C22).
  • alkynyl used either alone or in compound words, denotes straight chain or branched hydrocarbon residues containing at least one carbon to carbon triple bond. Prefixes such as “C2-C20” are used to denote the number of carbon atoms within the alkenyl group (from 2 to 20 in this case).
  • optionally substituted typically refers to where a hydrogen atom on a group has been substituted with a non-hydrogen group. Any optionally substituted group may bear one, two, three or more optional substituents.
  • non-naturally occurring amino acids include any compound with both amino and carboxyl functionality, derivatives thereof, or derivatives of a naturally occurring amino acid. These amino acids form part of the peptide chain through bonding via their amino and carboxyl groups. Alternatively, these derivatives may bond with other natural or non-naturally occurring amino acids to form a non-peptidyl linkage.
  • the compounds of the present invention may exist in one or more stereoisomeric forms (e.g. diastereomers).
  • the present invention includes within its scope all of these stereoisomeric forms either isolated (in, for example, enantiomeric isolation), or in combination (including racemic mixtures and diastereomic mixtures).
  • the present invention contemplates the use of amino acids in both L and D forms, including the use of amino acids independently selected from L and D forms, for example, where the peptide comprises two cyclohexylalanine residues, each residue may have the same, or opposite, absolute stereochemistry.
  • the invention thus also relates to compounds in substantially pure stereoisomeric form with respect to the asymmetric centres of the amino acid residues, e.g., greater than about 90% de, such as about 95% to 97% de, or greater than 99% de, as well as mixtures, including racemic mixtures, thereof.
  • diastereomers may be prepared by asymmetric synthesis, for example, using chiral intermediates, or mixtures may be resolved by conventional methods, e.g., chromatography, or use of a resolving agent.
  • each formula includes compounds having the indicated structure, including the hydrated as well as the non- hydrated forms.
  • Known solid or solution phase techniques may be used in the synthesis of the peptides of the present invention, such as coupling of the N- or C-terminus to a solid support (typically a resin) followed by step-wise synthesis of the linear peptide.
  • a solid support typically a resin
  • Protecting group chemistries for the protection of amino acid residues, including side chains are well known in the art and may be found, for example, in: Theodora W. Greene and Peter G. M. Wuts, Protecting Groups in Organic Synthesis (Third Edition, John Wiley & Sons, Inc, 1999), the entire contents of which is incorporated herein by reference.
  • Methods for the preparation of conjugates as described herein will be apparent to those skilled in the art and will comprise the steps of a) defining the distance between (a) the C- terminus of the formylated peptide and a binding and/or interaction site of the glycopeptide antibiotic agent; b) selecting a linker which is capable of spanning the distance as defined in (a); and c) bonding the formylated peptide and the glycopeptide antibiotic agent via the linker as selected in (b).
  • a linker comprises the selection of linkers known in the art as well as the generation and use of novel linkers, for example, by molecular modelling and corresponding synthesis or further methods known in the art.
  • the term "spanning" as used herein with reference to step b) refers to the length of the linker selected to place the glycopeptide antibiotic agent at the correct locus on a bacteria while enabling the formylated peptide to function in its role as a chemoattractant.
  • linker moiety is to connect the glycopeptide antibiotic agent to the formylated peptide in order to allow the glycopeptide antibiotic agent to interact with the bacteria while the formylated peptide acts as a chemoattractant.
  • the glycopeptide antibiotic agent and the linker will contain functional groups allowing for the two to be covalently bonded.
  • the nature of the functional group of the glycopeptide antibiotic agent is in no way limited and may include, for example, an amine group that forms an amide bond with the linker, or a hydroxyl or carboxylic acid group that forms and ether or ester bond with the linker.
  • vancomycin has three potential sites for connection to the linker as illustrated below, being the primary amine on the vancomycin sugar (V-linked, i), the secondary methyl amine (N-linked, ii) and the carboxylic acid (C-linked, iii). Coupling the linker to the primary amine is straightforward and high yielding. As the primary amine is more reactive, it is required to be protected with a protecting group such as a Boc- protecting group before the linker can be coupled to secondary amine (ii). Similarly, the carboxylic acid cannot be reacted directly, as activating the acid will cause it to react with the amine groups on vancomycin. A Boc protecting strategy may be used to first mask the primary amine and then functionalise the other two positions.
  • a protecting group such as a Boc- protecting group
  • the methylated amine can be functionalised, for example, by coupling with 5-azidopentanoic acid and the carboxylic acid can be functionalised by coupling with 3-azido-l- propanamine, after which they may be deprotected.
  • the skilled addressee will also understand that selection of the functional group at the end of the linker that connects with the formylated peptide will be dictated primarily by any available functional groups on the formylated peptide of choice. As an example, if the formylated peptide comprises a free amine or carboxylic acid group, it is envisaged that the functional group of the linker will comprise a complementary carboxylic acid or amine to form an amide bond.
  • treatment may include alleviating or ameliorating the symptoms, diseases or conditions associated with the microbial infection being treated, including reducing the severity and/or frequency of the microbial infection.
  • prevention may include preventing or delaying the onset of, inhibiting the progression of, or halting or reversing altogether the onset or progression of the particular symptoms, disease or condition associated with a microbial infection.
  • the bacterial infection may be caused by one or more species selected from one or more of the Gram-positive bacterial genera: Actinobacteria, Bacillus, Clostridium, Corynebacterium, Enterococcus, Listeria; Nocardia, Staphylococcus, and Streptococcus. Specific examples include, but are not limited to, Listeria monocytogenes and Staphylococcus aureus.
  • the invention provides a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate according to the invention, or a pharmaceutically acceptable salt thereof.
  • the invention provides a conjugate according to the invention, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a bacterial infection in a subject in need thereof.
  • the invention provides use of a conjugate according to the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a bacterial infection in a subject in need thereof.
  • the bacterial infection is a Gram-positive bacterial infection from the genera Actinobacteria; Bacillus, Clostridium, Corynebacterium, Enterococcus, Listeria, Nocardia, Staphylococcus, or Streptococcus.
  • the Grampositive bacterial infection is caused by methicillin-resistant Staphylococcus aureus.
  • the bacterial infection may be caused by one or more species selected from one or more of the Gram-negative bacterial genera: Acinetobacter; Actinobacillus; Bartonella', Bordetella', Brucella', Burkholderia', Campylobacter, Cyanobacteria', Enterobacter, Erwinia', Escherichia', Francisella', Helicobacter, Hemophilus', Klebsiella', Legionella', Moraxella', Morganella; Mycobacterium', Neisseria', Pasteurella', Proteus', Providencia', Pseudomonas', Salmonella', Serratia', Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia. Specific examples include, but are not limited to, infections caused by Helicobacter pylori, uropathogenic Escherichia coli, Mycobacterium tuberculosis and Ps
  • subject is intended to include organisms such as mammals, e.g. humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human, e.g. a human suffering from, at risk of suffering from, or potentially capable of suffering from a bacterial infection.
  • the subject is a cell.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a conjugate as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
  • composition is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.
  • conjugates as hereinbefore described, or pharmaceutically acceptable salts thereof may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the conjugate is within the scope of the invention.
  • the conjugate could be administered with one or more therapeutic agents in combination.
  • the combination may allow for separate, sequential or simultaneous administration of the conjugate as hereinbefore described with the other active ingredient(s).
  • the combination may be provided in the form of a pharmaceutical composition.
  • combination refers to a composition or kit of parts where the combination partners as defined above can be dosed dependently or independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points.
  • the combination partners can then be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the ratio of the total amounts of the combination partners to be administered in the combination can be varied, e.g., in order to cope with the needs of a patient sub -population to be treated or the needs of the single patient which different needs can be due to age, sex, body weight, etc. of the patient.
  • the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art.
  • care should be taken to ensure that the activity of the conjugate is not destroyed in the process and that the conjugate is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the conjugate by means known in the art, such as, for example, micro encapsulation or coating (such as the use of enteric coating).
  • the route of administration chosen should be such that the conjugate reaches its site of action.
  • Those skilled in the art may readily determine appropriate formulations for the conjugates of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.
  • the conjugates as hereinbefore described, or pharmaceutically acceptable salt thereof may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery.
  • the pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi.
  • the solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the conjugate, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • agents to adjust osmolarity for example, sugars or sodium chloride.
  • the formulation for injection will be isotonic with blood.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.
  • Sterile injectable solutions are prepared by incorporating the active conjugate in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.
  • compositions include oral and enteral formulations of the present invention, in which the active conjugate may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard- or soft-shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active conjugate may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the amount of active conjugate in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, com starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active conjugate may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active compound to specific regions of the gut.
  • Liquid formulations may also be administered enterally via a stomach or oesophageal tube.
  • Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the conjugates of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like.
  • the present invention also extends to any other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions.
  • topical application such as creams, lotions and gels
  • compositions suitable for inhalation or intranasal delivery for example solutions, dry powders, suspensions or emulsions.
  • the compounds of the present invention may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases.
  • a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases.
  • the conjugates may also be administered using a nebuliser.
  • Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 pg to about 200 mg. Expressed in proportions, the active compound may be present in concentrations ranging from about 0.25 pg to about 200 mg/mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the term "effective amount" refers to an amount of conjugate which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur once, or at intervals of minutes or hours, or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. A typical dosage is in the range of 1 pg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
  • Fmoc -protected L-amino acids were purchased from Sigma-Aldrich (MO, USA), except for Fmoc-Met(0)-OH, Fmoc-Met(0 2 )-OH, Fmoc-Nle-OH, Fmoc-Nva-OH, Fmoc-a-t-butyl-Gly-OH (Fmoc-t-Feu-OH), Fmoc-Phe(4- F)-OH, Fmoc-Phe(4-NH 2 ), Fmoc-Phe(4-Cl)-OH, Fmoc-Phe(4-CN)-OH and Fmoc-NH- (PEG)2-OH, which were purchased from Merck (NJ, USA); Fmoc-Met(0)-OH, Fmoc-Met(0 2 )-OH, Fmoc-Nle-OH, Fmoc-Nva-OH, Fmoc-a-t-butyl-Gly-OH (
  • Peptides were completed using a combine/split method where lanterns were mixed for common steps such as washing, amino acid coupling and deprotection steps and split for coupling specific amino acids when required.
  • a 20 mL glass vial polystyrene lanterns with Fmoc-protected Rink amide linker were added (loading 37 pmol/lantern). Lanterns were swelled in DCM for 30 min.
  • Fmoc deprotection 20% piperidine in DMF (20 min), followed by 5 x 3 min DMF washes.
  • Rink amide NovaPEG resin (Novabiochem) (681.8 mg, loading 0.3 mmol) was placed in a syringe with a filter frit and swelled in DCM for 30 min, then flushed with DMF (3 x 30 sec).
  • Fmoc-protected propargylglycine (Pra) (2 eq) was activated with HCTU (1.95 eq) and DIPEA (4 eq) for 5 min, and then added to the resin. Coupling was completed overnight, followed by DMF washing (3 x 30 sec). The resin was then dried with diethyl ether and divided into three batches for linkers a, b and c (La, Lb and Lc).
  • the Fmoc protecting group of the second batch Lb and third batch Lc was removed with 20% piperidine in DMF for 30 min, followed by DMF washing (4 x 30 sec). Two eq Fmoc- NH-(PEG)2-C0 2 H (13 atoms, see below) was then activated with HCTU (1.95 eq) and DIPEA (4 eq) for 5 min and added to the resin of batch two and three. Fmoc-NH-PEG(2)-C0 2 H (13 atoms)
  • 5-Azidopentanoic acid (1.1 eq) was pre-activated with COMU (1.1 eq) and TEA (2 eq) in DMF for 5 min. This solution was added onto vancomycin HC1 (1 eq) dissolved in DMF at -20-50 mM, stirred overnight and analyzed by FCMS. The product was precipitated in diethyl ether and washed twice with diethyl ether, giving crude in -85-98% yield.
  • FC analysis allowed to determine that the ratio between 4 compounds, namely vancomycin, azidopentanoyl on the N-terminal, azidopentanoyl on the vancosamine moiety and the double addition that were all isolated by RP-HPFC preparative (30%/ 40%/ 10%/ 20%).
  • the sugars from Teicoplanin were removed using a mixture of HC1/ isopropanol to enable the selective modification of either N-terminal amino group or the C-terminal carboxylic acid group (Scheme 3).
  • the N-terminal azidopentyl-teicoplanin aglycone was obtained after the coupling of 5-azidopentanoic acid with DIC and Oxyma to the teicoplanin aglycone.
  • the C-terminal azidopropyl-teicoplanin aglycone was obtained in three steps involving the protection the N-terminal amine group with a Boc group, the C-terminal modification with 3-azidopropyl amine and the removal of the Boc group with a solution of TFA/ DCM.
  • Solid phase peptide synthesis was used assemble the peptide starting from Wang resin that was acylated with Fmoc-L-Arg(Pbf)-OH (Scheme 4). Peptide elongation was performed using a solution of 20% piperidine for Fmoc removal and DIC/ Oxyma for Fmoc-amino acid or rhodamine B coupling. The completed peptide was cleaved from the resin by TFA cleavage.
  • Scheme 4 Preparation of Rhodamine B-PB10 peptide - inhibitor of FRP2 by solid phase peptide synthesis.
  • HSA Human serum albumin
  • HBSS Hank’s Balanced Salt solution
  • RPMI Roswell Park Memorial Institute medium
  • HSA Human serum albumin
  • HBSS Hank’s Balanced Salt solution
  • RPMI Roswell Park Memorial Institute medium
  • MA ThermoFischer Scientific
  • MA ThermoFischer Scientific
  • MA ThermoFischer Scientific
  • MA ThermoFischer Scientific
  • Bacteria were streak plated from glycerol stocks onto BHI agar plates and grown at 37 °C for 16 h.
  • An overnight culture was prepared by inoculating 2-3 colonies into BHI broth at 37 °C for 16 h with shaking.
  • the number of colony forming units (CFUs) was determined by plating 10-fold serial dilution of overnight culture onto BHI agar plates in duplicate. Absorbance at 600 nm was determined for a 1:10 dilution of overnight culture and the CFUs per unit of absorbance was calculated.
  • Vancomycin HC1 (1 eq), fMFFK-NH2 (FP18, 1 eq) and vancomycin conjugated to FP1 via 4 PEG units and a triazolo linkage (FPl-Ld-C-Van, 1 eq) were dissolved in DMF and the pH 8 was obtained by the addition of TEA. Then, BODIPY® FL NHS Ester (l.leq) dissolved in DMF was added, the reaction was monitored by LCMS and the product was purified directly by preparative RP-HPLC.
  • BODIPY was also conjugated to vancomycin via the C terminus of the antibiotic and to a formylated peptide conjugated to vancomycin via linker a (La) to ensure the BODIPY attachment location was not altering binding profiles observed.
  • Labelling of vancomycin was achieved following a three step protocol involving the preparation of azido-propyl vancomycin from vancomycin and BODIPY-alkyne from BODIPY® FL NHS Ester (Scheme 5). The resulting click chemistry between the BODIPY-alkyne and the azido-propyl vancomycin afforded the BODIPY labelled vancomycin.
  • S. aureus strains (MSS A, ATCC 29213; MRS A, A8090; VISA, A8094) were grown to mid-exponential phase and incubated with 16 mM of either the BODIPY-fluorescein- labelled vancomycin or the FPl-Ld-C-Van conjugate labelled with BODIPY for 30 min at room temperature. The cells were collected by centrifugation and washed to remove excess unincorporated compound, followed by resuspension in fresh MHB broth.
  • STED stimulated emission depletion
  • the cells were imaged using a Zeiss LSM 980 Airyscan 2 (Carl-Zeiss, Jena, Germany) with a 63x oil C-PlanApo 1.4NA objective using the optimal super-resolution mode.
  • Three lines were drawn across the cell that intersected the cell wall and the septum. The intensity of fluorescence along these lines was plotted and the peaks determined. These peaks in fluorescence corresponded to the walls and the septum of the cell.
  • the ratio of fluorescence at the septum compared to the wall was then calculated and plotted in GraphPad Prism. Image analysis was performed an at least 20 cells per biological replicate and at least 3 biological replicates were scored for each treatment.
  • a 20 pL sample was diluted with 20 pL trypan blue and cells were counted using in a haemocytometer. Cell viability was determined by trypan blue exclusion. Neutrophils were only used if purity and viability was > 98%.
  • the chemotaxis assay was performed in a 96-transwell plate with 3pm pores (Coming).
  • the plate was prepared by adding 200 pL of peptide in chemotaxis buffer (4 mM L- glutamine, 0.5% HSA, 49% RPMI, 49% HBSS) into the bottom receiving wells at a final concentration of 1, 10, 100 and 1000 nM (0.02% DMSO).
  • a negative control of 0.02% DMSO was used.
  • the top of the transwell plate was loaded with 200,000 neutrophils in 75 pL chemotaxis buffer. The plate was incubated in the dark at 37 °C with 5% CO2 for
  • the absorbance at 405 nm was determined using a microplate reader (Clariostar plate reader, BMG). Absorbance readings were converted to number of neutrophils using a standard curve of known neutrophil numbers. All peptides were tested in duplicate with 3-4 biological replicates, ensuring different donors each time.
  • Human neutrophils displayed a variability of response to formylated peptides between individual donors. This variability was taken into account by determining the percentage of neutrophil recruitment relative to the recruitment observed to the fMLFG peptide at 100 nM from the same neutrophil donor. To compare data between donors, the positive control of 100 nM fMLFG (FP1) was set as 100% and all other conditions calculated relative to this. Data was plotted using GraphPad Prism 8 software.
  • PDMS polydimethylsiloxane, Fischer Scientific, Fair Lawn, NJ microfluidic devices were generated through soft lithography from silicon wafers fabricated from chrome masks designed previously (Ellett, F. el al. Lab on a Chip, 2019, 19, 1205-1216). Fabrication and use of this microfluidic device for monitoring neutrophil migration and killing of live S. aureus (SH1000-GFP) or pHrodo Red S. aureus BioParticles® (Life Technologies) in cell culture medium (IMDM + 10% FBS) has been outlined in detail by the designers (Ellett el al. 2019).
  • RhB-PBlO The fluorescently labelled FPR2 antagonist, RhB-PBlO, was used to determine the interaction of the formylated peptides with FPR2 of human neutrophils.
  • RhB-PBlO (15uM) was preincubated with human neutrophils (5x10 ⁇ 6neutrophils/mL) in Bis-Tris buffer pH6 for lh, washed twice in Bis-Tris buffer pH6, before incubating with 8uM of peptide for 5min. Neutrophils were washed, and then resuspended in Bis Tris buffer.
  • Rhodamine B levels of the samples were determined using microplate reader (Clariostar plate reader, BMG) with ex 550 nm, em 585 nm.
  • mice Eight-week-old female Balb/c mice were anaesthetised with isoflurane, before intranasal inhalation of 10 7 cfu S. aureus, in 50 ⁇ L of PBS.
  • One-hour post infection (hpi) mice were treated intranasally with 50 pL of vancomycin (0.2 mg/50 pL) or equivalent molar amount of Fmlfg (FP1), FP9-La-C-Van or vehicle control (5% DMSO in PBS).
  • Mice were humanely euthanized by CO2 inhalation at 12 hpi and lung bacterial density was assessed from four mice per treatment group.
  • Lungs were homogenised and underwent serial dilution and plating onto solid media for CFU enumeration. Significance was determined using a Mann-Whitney test with p ⁇ 0.02 being significant and calculated using GraphPad Prism 8 software.
  • the formylated peptide-vancomycin conjugates were tested for antibacterial activity against S. aureus. Two strains were used: A8090, a vancomycin-sensitive S. aureus clinical isolate, and A8094, a vancomycin-intermediate S. aureus isolate from the same patient.
  • the inhibition of bacterial growth was tested over a range of concentrations of compound using a broth microdilution plate assay. The amount of bacterial growth is reported as a percentage of bacterial growth in absence of compound (negative control). The inhibition is then compared to vancomycin to characterise the effect of conjugation and linker length on the antibacterial activity of the conjugated vancomycin. The experiment was performed in three biological replicates, each time in triplicate.
  • IC50 concentration of 50% growth inhibition
  • MIC minimum inhibitory concentration
  • conjugation of formyl peptide at this site is not a viable strategy for direct killing of S. aureus.
  • these compounds by extension are also likely to have much lower affinity for the bacterial cell wall and thus may not be capable of generating the chemoattractant gradient necessary for recruitment of neutrophils.
  • the peptide backbone of vancomycin should not be affected by modification of the sugar moiety.
  • a reduction in antimicrobial activity was observed by linking to through the vancosamine primary amine. This reduction may be attributed to the linkage affecting the dimerization that occurs through these sugars and is important for activity.
  • Vancomycin is produced by Amycolatopsis orientalis, and logically, the metabolic expense of the biosynthesis of the sugars should be justified by serving an important function that provides the bacterium with a survival advantage. NMR evidence has demonstrated that the sugars on vancomycin are necessary for dimerization to occur (Gerhard, U.; et ah, J. Am. Chem. Soc. 1993, 115 (1), 232-237).
  • vancomycin dimerization is enhanced when bound to the cell wall, and its binding to the cell wall is enhanced by dimerization (Williams, D. H.; Maguire, A. J.; Tsuzuki, W.; Westwell, Science 1998, 280 (5364), 711). From the experiments, it is evident that conjugation of formyl peptide to the primary amine has blocked the sugar moiety, inhibiting the dimerization of vancomycin and therefore reducing its antibacterial activity.
  • Vancomycin is clinically not effective against VISA, which by definition are strains with MIC > 16 pg/ml (11 pM); thus, conjugates were only tested up to 30 pM.
  • vancomycin is ineffective at treating VISA and VRSA because of limited membrane penetration (Rybak, M. J., Clin. Infect. Dis. 2006, 42 (Supplement_l), S35- S39).
  • An additional issue with using high concentrations of vancomycin is its potential for nephrotoxicity and ototoxicity, especially when used in combination with other drugs (Rybak, M.; et al, Am. J. Health Syst. Pharm. 2008, 66 (1), 82).
  • alternative treatments are implemented for patients infected with S.
  • aureus isolates that have MIC > 2 pg/ml (1.38 pM). Therefore, these results show that the antibacterial activity alone of the conjugated compounds will not achieve positive clinical outcomes for treating MRSA or VISA.
  • the most critical feature of these compounds is their ability to recruit neutrophils to eliminate S. aureus. Generating the chemical gradient that leads the neutrophils to S. aureus is dependent on the ability of the compounds to bind to the cell wall. As these compounds have antibacterial activity against vancomycin-susceptible S. aureus (VSSA), they should be capable of binding to the cell wall in a similar fashion to vancomycin.
  • VSSA vancomycin-susceptible S. aureus
  • Conjugating FP1 has a slight detrimental effect to its chemotactic activity. Regardless, the compounds were still potent chemoattractants with similar activity to the parent peptide. Ignoring the effect of linker size, comparing conjugation at the three different sites: the vancosamine primary amine; methylated amine; or the carboxyl group, there is little difference in chemotaxis profiles. Thus, the reduction in chemotactic activity of the conjugates compared with fMLFG is merely a consequence of conjugation, and is not a result of the particular position of attachment of formyl peptide to vancomycin. It appears that the ability of the formyl peptide moiety to stimulate chemotaxis is much the same no matter where it is conjugated onto vancomycin.
  • a library was designed based upon the fMLFG (FP1) sequence by combinatorial peptide synthesis on lanterns ( Figure 4). Each lantern was tagged and common synthetic steps could be achieved following a split/mix to speed up the elaboration of the library. Each of the residues of this sequence were modified to a variety of different natural and non-natural amino acids ( Figure 4). This gave a library of formylated peptide covering a range of different hydrophobic and size profiles ( Figure 4 and Table 1). Using a transwell migration assay, the chemotaxis of neutrophils to these peptides was examined at different concentrations (1-1000 nM).
  • microbroth dilution assay it was also determined that the sequence of the formylated peptide attached to vancomycin did not have an effect on antimicrobial activity.
  • a two-fold serial dilution of the conjugates was tested from 30 to 0.46 uM against three strains of S. aureus- MSS A, MRS A, and VISA. Irrespective of the formylated peptide attached to vancomycin, the MIC against the strains of S. aureus tested remained the same (Table 4).
  • fMLF is described as a preferential activator of FPR1, and resulted in similar levels of RhB-PBlO fluorescence bound to human neutrophils as the no peptide control.
  • FPR binding of the formylated peptide was altered by conjugating to vancomycin.
  • the FP9-Van conjugate resulted in no loss of fluorescence suggesting it was not able to compete with RhB-PBlO binding to FPR2. This is in contrast to the FP9 peptide, which did reduce fluorescence and compete with RhB-PBlO binding.
  • Conjugate reduces inflammation and bacterial load in S. aureus mouse pneumonia
  • Conjugate recruits neutrophils to Gram-negative bacteria
  • Airy scan imaging was used to determine binding location of the C terminal BODIPY labelled vancomycin and formylated peptide linked C terminal to vancomycin with a variety of heat killed bacteria ( Figure 16). It was confirmed that compounds bind to the heat killed strains of MSS A, MRS A and VISA in the same pattern of binding as the live strains previously tested. No binding of the BODIPY labelled formylated peptide alone was observed to all of the heat killed bacteria tested. Both the BODIPY labelled conjugate and vancomycin were observed bound to the Gram-positive heat killed strains of Bacillus subtillus, and Listeria monocytogenes (Figure 16).
  • Gram-negative heat killed E.coli 0111:B4 was also bound by the BODIPY labelled compounds along with an avirulent strain of Mycobacterium tuberculosis H37 Ra (Invivogen) although to a lesser extent than the Gram-positive strains.
  • Particles were either used directly or washed twice with 2 volumes of PBS before respending in IMDM + 10% PBS. Samples were loaded into microfluidic devices and standard neutrophil migration assay performed. As can be seen from Figure 17, bacteria incubated with FP1 and washed have less recruitment compared to FPl-La-C-Van washed. For E. coli, Pseudomonas, and Mycobacterium the recruitment of washed FPl-La-C-Van is similar to the recruitment observed for unwashed FPl-La-C-Van.

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Abstract

The present invention relates to conjugates comprising a glycopeptide antibiotic agent and a chemotactic formylated peptide and their use in the treatment or prevention of bacterial infections. The invention also relates to use of the conjugates in the prevention or treatment of bacterial infections.

Description

Antibiotic conjugates
Field of the invention
The invention relates to conjugates comprising a glycopeptide antibiotic agent and a chemotactic formylated peptide and their use in the treatment or prevention of bacterial infections. The conjugates of the invention are particularly suited to the prevention or treatment of bacterial infections caused by Staphylococcus aureus.
Background of the invention Antibiotic resistance is an increasing threat worldwide with widespread antibiotic use. Some strains, such as methicillin-resistant Staphylococcus aureus (MRSA), have developed multidrug resistance (MDR), raising significant concern in the medical community. In the majority of the world, over 20% of S. aureus strains are methicillin- resistant (World Health Organization, Antimicrobial resistance: Global report on surveillance ; June, 2014; pp 19-21). S. aureus is a well-adapted human pathogen, which expresses many virulence factors that allow it to colonise tissues and evade and resist the immune system. With added antibiotic resistance S. aureus represents a deadly threat to hospital patients, who are immunocompromised or present open wounds. Recently, the World Health Organization listed MRSA, vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) as high priority targets for the development of new antibiotics due to the serious nature of these threats to modern medicine.
There is an urgency to find novel treatments to overcome bacterial resistance to antibiotics. Vancomycin is often used as the last resort treatment for MRSA (Liu, C.; et al., Clin. Infect. Dis. 2011, 52 (3), el8-e55), as these bacteria are resistant to b-lactam antibiotics. Successful control of antibiotic resistant bacteria is dependent on a variety of strategies, including identifying new targets, deepening understanding of underlying resistance mechanisms, optimising existing drugs, rational drug design and combinatorial biology (Hughes, D. Nat. Rev. Genet. 2003, 4 (6), 432-41). Since MRSA strains often develop reduced susceptibility to vancomycin, there is a need to develop new treatment strategies against this pathogen that causes -11,000 deaths a year in the US alone (Dantes, R.; et ah, JAMA Intern. Med. 2013, 173 (21), 1970-1978).
The body’s innate immune response against bacterial pathogens like S. aureus is the primary route for the clearance of such infections. Neutrophils are the major effectors of the innate immune response against infection and comprise approximately 50-70% of all leukocytes in the bloodstream (Mocsai, A., J. Exp. Med. 2013, 210 (7), 1283). Individuals with low neutrophil count (neutropenia) are more susceptible to infection as a consequence (Gibson, C.; Berliner, N., Blood 2014, 124 (8), 1251-1258). Neutrophils are well-equipped to kill bacterial pathogens directly and have three main ways of doing so: phagocytosis, degranulation and neutrophil extracellular traps (NETs).
When neutrophils phagocytose or ingest bacteria it results in the formation of a phagosome into which they secrete reactive oxygen species (ROS) and enzymes to break down the bacteria. They produce ROS through an oxidative burst: NADPH oxidase transfers electrons to oxygen to generate the superoxide anion (O2 ), which then converted into other highly toxic reactive species, such as hydrogen peroxide (H2O2) and hypochlorous acid (HOC1) (Dale, D. C.; Boxer, L.; Liles, W. C., Blood 2008, 112 (4), 935-945).
In a process known as degranulation, neutrophils release granules containing an array of proteolytic enzymes and membrane -permeabilising proteins and peptides, including elastase, lysozyme, defensin and bactericidal/permeability-increasing protein (BPI) (Cowland, J. B.; Borregaard, N., Immunol. Rev. 2016, 273 (1), 11-28). These granules also contain NADPH oxidase and myeloperoxidase (MPO), which assist in generating ROS.
Neutrophils are also capable of killing and inhibiting the growth of bacteria by forming NETs. NETs are comprised of antimicrobial peptides and enzymes bound to a mesh of DNA, and are formed when neutrophils are stimulated by a range of factors including bacterial pathogens and ROS (Arazna, M.; Pruchniak, M. P.; Demkow, U., Respir. Physiol. Neurobiol. 2013, 187 (1), 74-77). This additional anti-bacterial mechanism can immobilise and kill bacteria that cannot be phagocytosed. Neutrophils patrol the body through the bloodstream, and are recruited from circulation to an infected site by chemical signals. Early on in the infection, immune cells that patrol the peripheral tissues such as macrophages, mast cells and dendritic cells are activated by the presence of bacteria and release chemokines to recruit neutrophils (Sokol, C. L.; Luster, A. D., Cold Spring Harb. Perspect. Biol. 2015, 7 (5), 1-20). Chemokines are signalling proteins secreted by cells that induce chemotaxis, i.e. movement in the direction of an increasing chemical gradient. Endogenous signals released by the host as well as some foreign signals induce chemotaxis in neutrophils. These molecular signals are detected by G-protein coupled receptors (GPCRs), which upon binding the ligand trigger downstream signalling pathways, activating several responses including actin rearrangement and adhesion (Dorward, D. A.; et al., Am. J. Pathol. 2015, 185 (5), 1172-1184). Once stimulated, neutrophils are targeted to the source of chemoattractant and release other signalling molecules such as leukotriene B4 (LTB4), signalling to other neutrophils and leukocytes to migrate from the bloodstream. This sets off a cascade that results in a significant increase in the recruitment of immune cells to the site of infection (Afonso, P. V.; et al., Dev. Cell 2012, 22 (5), 1079-1091). The recruitment of neutrophils via chemotaxis is vital for the body to mount a successful immune response against virulent bacteria such as S. aureus.
Just as the human immune system has developed efficient defences against S. aureus, through co-evolution S. aureus has also adapted to efficiently evade the immune system (Thammavongsa, V.; et al., Nat. Rev. Microbiol. 2015, 13, 529-543). S. aureus can also inhibit the key neutrophil functions of killing by ROS, phagocytosis and chemotaxis.
S. aureus can reduce the damage of ROS by expression of superoxide dismutase and staphyloxanthin, the yellow carotenoid produced by S. aureus responsible for its golden colour (Liu, G. Y.; et al., J. Exp. Med. 2005, 202 (2), 209-215). Staphyloxanthin acts as an antioxidant, neutralising the ROS produced in the phagosome of the neutrophil. The phagocytosis of bacteria by neutrophils requires recognition of pathogen-associated molecular patterns (PAMPs) on the surface of the bacteria by pattern recognition receptors (PRRs). Phagocytosis is greatly enhanced when opsonins such as IgG antibodies and complement components are bound to the bacteria. The polysaccharide capsule surrounding most S. aureus strains can prevent the binding of opsonins (Foster, T. J., Nat. Rev. Microbiol. 2005, 3 (12), 948-958). A surface protein on S. aureus, protein A, can bind antibodies via their Fc domain (the region that is recognised by neutrophils), which leads to reduced phagocytosis. Furthermore, S. aureus can express other cell wall- anchored proteins that promote adhesion to the extracellular matrix and form biofilms. These include clumping factor A (clfA), collagen adhesin (Cna) and biofilm-associated protein (Bap) (Foster, T. J.; et al., Nat. Rev. Microbiol. 2014, 12 (1), 49-62). These virulence factors offer protection from phagocytosis, thereby inhibiting the ability of neutrophils to recognise and kill S. aureus.
Over 60% of S. aureus strains are known to produce a virulence factor known as chemotaxis inhibitor protein of S. aureus (CHIPS), that binds to FPR and C5a receptor, and antagonise their ability to induce chemotaxis (Postma, B.; et al., J. Immunol. 2004, 172 (11), 6994-7001). Similarly, around 59% of strains produce a related protein known as formyl peptide receptor-like 1 inhibitory protein (FLIPr) that antagonise the ability of FPR2 to induce chemotaxis (Prat, C.; et al., J. Immunol. 2006, 177 (11), 8017).
With so many ways of evading immune detection, it is unsurprising that S. aureus is a huge problem in the clinic and community. Infected individuals often rely on effective antibiotic therapy for successful treatment. However, with the emergence of multi-drug resistance, this antibiotic treatment strategy is no longer always useful. There is a critical need for novel treatment strategies to combat antibiotic resistant bacteria.
Summary of the invention
New conjugates and methods are provided for the treatment or prevention of bacterial infections. Accordingly, in one aspect, the present invention provides a conjugate of formula (I):
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof, wherein GPA is a glycopeptide antibiotic agent;
L is a linker moiety; and
FP is a chemotactic formylated peptide.
In another aspect, the invention provides a formylated peptide, or pharmaceutically acceptable salt thereof, selected from:
Figure imgf000006_0002
Figure imgf000007_0001
Figure imgf000008_0001
wherein
R1 represents a side chain of an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline;
R2 represents a side chain of an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine;
R3 represents a side chain of an amino acid selected from tyrosine, aspartic acid, 4- fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
R4 represents a side chain of an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
R4a represents a side chain of an amino acid selected from leucine, arginine, glutamic acid, glutamine, histidine, serine, or phenylalanine; and
R5 is selected from NH2, OH, or SH.
The invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a conjugate of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
In another aspect, the invention provides a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate of the invention, or a pharmaceutically acceptable salt thereof.
A further aspect the invention provides a conjugate of the invention, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a bacterial infection in a subject in need thereof.
Another aspect the invention provides use of a conjugate according to the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a bacterial infection in a subject in need thereof.
These and other aspects of the present invention will become more apparent to the skilled addressee upon reading the following detailed description in connection with the accompanying examples and claims.
Brief description of the drawings
Figure 1: Antibacterial activity of formylated peptide-vancomycin conjugates.
Antibacterial activity of conjugates against MRSA (A) or VISA (B) was determined for the conjugates of formylated peptide FP1 linked to vancomycin at the three sites; vancosamine primary amine (V-linked), methylated amine (N-linked); or the carboxyl group (C-linked); with 3 different length PEG linkers of 0 (La), 3 (Lb), or 6 (Lc) repeats. Growth of a paired clinical isolate of S. aureus - MRSA and VISA strains were determined using microbroth dilution assay. Growth was calculated relative to the no protein control. Error bars are SEM, n=3 biological replicates tested in technical triplicate each time.
Figure 2: Chemotactic activity of the conjugates of formylated peptide (fMLFG, FP1) linked to vancomycin at the three sites; vancosamine primary amine (V-linked), methylated amine (N-linked); or the carboxyl group (C-linked); with 3 different length PEG linkers of 0 (La), 3 (Lb), or 6 (Lc) repeats. Chemotaxis was calculated relative to a no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor. Error bars are SEM, n=3 different biological donors tested in technical triplicate.
Figure 3: Comparison of binding of vancomycin and conjugate binding to 3 strains of S. aureus. Vancomycin or the formylated peptide conjugated to vancomycin through the C terminus with a 4 repeat PEG linker (FPl-Ld-C-Van) were fluorescently labelled with BODIPY and their binding to the S. aureus was imaged by STED microscopy. Scale bar is lμm. Figure 4: AiryScan imaging of S. aureus strains (MSS A, A; MRS A, B; VISA, C) incubated with either BODIPY C-terminally labelled vancomycin (Vancomycin) or BODIPY labelled formylated peptide linked to vancomycin (fPep=Van) in the addition to wheat germ agglutinin (WGA) and Hoechst. The ratio of fluorescence observed at the septum compared to the cell wall was plotted for the three strains, for both the BODIPY labelled peptides, which bound to the septum; and the WGA that showed little septum binding.
Figure 5: The sequence of the formylated peptide effects neutrophil recruitment. (A) Schematic of the process of creating and testing a library of formylated peptides based on the fMLFG (FP1) sequence. The library of formylated peptides were grouped into 6 profiles (line graphs, B-F), based on the concentration of the formylated peptide that resulted in the greatest recruitment of neutrophils in a transwell assay. This was a peak observed at: 100 nM (B), at both 10 and 100 nM (C), at 10 nM (D), at 100 and 1000 nM (E), at 1000 nM (F). Chemotaxis was calculated relative to the no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor, n>3, error bars are SEM. Dotted line on graphs represents the media only control recruitment level.
Figure 6: The formylated peptide sequence conjugated to vancomycin effects neutrophil recruitment Representative formylated peptides from each of the 6 formylated peptide library profiles were linked to the C-terminus of vancomycin and retested for the ability to recruit neutrophils using the same transwell method (solid bars represent conjugate). This was a peak observed at: 100 nM (A), at both 10 and 100 nM (B), at 10 nM (C), at 100 and 1000 nM (D), at 1000 nM (E). Chemotaxis was calculated relative to the no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor, n>3, error bars are SEM. Dotted line on graphs represents the media only control recruitment level.
Figure 7: Peptides that resulted in no recruitment of human neutrophils in a transwell assay at 1, 10, 100, and 1000 nM. Chemotaxis was calculated relative to the no protein control and 100% chemotaxis set as the neutrophil recruitment observed to FP1 at 100 nM for each donor, n>3, error bars are SEM. Dotted line is the media only control recruitment level.
Figure 8: In silico comparison of LogP (A) and solvent accessible surface area I SASA (B) of formylated peptide FP1 compared to FP2 to FP24.
Figure 9: Infection on a chip microfluidics used to monitor neutrophil migration and phagocytosis. (A) The infection-on-a-chip microfluidic device used to monitor neutrophil migration to compound and phagocytosis of S. aureus over time present in a 6 well plate. (B) Time course of neutrophils migrating into the microchamber containing S. aureus bioparticles labelled with pHrodo, in the presence of free formylated peptide (FP1 or FP9) or conjugated (FPl-La-C-Van or FP9-La-C-Van) at 1000 nM. Images are representative of four donors, scale bar is 10 pm.
Figure 10: Finking a formylated peptide to vancomycin enhances phagocytosis activity of neutrophils. The infection-on-a-chip microfluidic device was used to monitor neutrophil migration and phagocytosis over time. (A) The percentage of neutrophils recruited into the microchamber in the presence of conjugated (triangles, FPl-La-C-Van; FP9-La-C-Van) or free formylated peptide (circles, FP1; FP9, 1000 nM) increased over time. Data is the average of four donors, with error bars of SEM. (B) Area of pHrodo fluorescence increased over time when conjugated (triangles, FPl-La-C-Van; FP9-La-C-Van) or free formylated peptide is present (circles, FP1; FP9, 1000 nM). Data is the average of experiments conducted with four different donors, with error bars of SEM. (C) The sequence of the formylated peptide (FP1; FP9; FP10; FP16) effects efficiency of neutrophil phagocytosis as determined by the pHrodo fluorescence per number of neutrophils recruited per egg at 2 h. Error bars are SEM, n=4 different biological donors. Data from each donor is linked by dotted lines, with donors also having an effect on if enhanced phagocytosis was observed. Figure 11 : Graphical representation of competition binding to human neutrophils between the FPR2 antagonist RhB-PBlO and different formylated peptides and conjugates. The formylated peptide fMLF binds preferentially to FPR1 and was used as a negative control, while the formylated peptides fMVIL which binds preferentially to FPR2 was used as a positive control. The peptides fMLFG (FP1) and fMChaFG (FP9) and their corresponding conjugates were examined for their ability to compete off RhB-PBlO binding to neutrophils.
Figure 12: Graphical representation of recruitment of human neutrophils to the teicoplanin aglycone conjugates. A Transwell assay was used to measure the human neutrophil recruitment to the fMLFK (fPep) formylated peptide linked to either the N terminus of teicoplanin aglycon (Teio-N-fPep) or c terminus (Teico-C-fPep). Error bars are SD, results from one donor.
Figure 13: The conjugated formylated peptide to vancomycin reduces bacterial load and a mouse pneumonia model. (A) Eight-week-old female mice infected by intranasal inhalation of 107cfu S. aureus to induce pneumonia. One -hour post infection (hpi) mice were given intranasal therapy at 0.2 mg/mouse equivalent of vancomycin (van, vancomycin; FP1; or FP9-La-C-Van) or vehicle control (control). The lungs where collected 12 hpi. (B) Bacterial load in the lung tissue of four mice per treatment group was determined. The formylated peptide-van conjugate FP9-La-C-Van resulted in 2-fold lower bacterial load compared to vancomycin (van) alone (*p<0.02, n=4/group)
Figure 14: Conjugate treatment reduces inflammation and alveoli structure is retained in mouse pneumonia model. Eight-week-old female mice were infected by intranasal inhalation of 107cfu S. aureus to induce pneumonia. One -hour post infection (hpi) mice were given intranasal therapy at 0.2 mg/mouse equivalent of vancomycin (Van) or vehicle control (control). The lungs where collected 12 hpi and lung histology performed with haemotoxylin and eosin staining for one mouse per treatment. The formylated peptide FP1 or the conjugate FP9-La-C-Van displayed reduced infiltration of innate immune cells into the alveoli. Black arrows indicate neutrophils, white arrows proteinaceous fluid, and grey arrows indicate cocci bacteria.
Figure 15: Conjugate treatment reduces immune cell infiltrating and alveoli structure is retained in mouse pneumonia model. Eight-week-old female mice were infected by intranasal inhalation of 107cfu S. aureus to induce pneumonia. One-hour post infection (hpi) mice were given intranasal therapy at 0.2 mg/mouse equivalent of vancomycin (Van) or vehicle control (control). The lungs where collected 12 hpi and lung histology performed with haematoxylin and eosin staining for one mouse per treatment. 6 fields of view (FOV) from the outer lobes of the lungs were analysed for the alveoli area (A) or the number of nuclei (B) present. The conjugate FP9-La-C-Van displayed similar infiltration of innate immune cells into the alveoli and the area of the alveoli as the uninfected normal lung (dotted line). Data is the average of 6 FOV from one mouse lung, with error bars of SEM.
Figure 16: Binding patterns of BODIPY labelled Vancomycin or the formylated peptide conjugated to vancomycin (fPep=van) to variety of heat killed bacterial strains. Bodipy fluorescence was universally set across all images to be able to compare relative levels. Images were captured using Zeiss LSM 980 Airyscan 2 (Carl-Zeiss, Jena, Germany). Scale bar 5 microns.
Figure 17: Graphical representation of the percentage of neutrophils recruited in the presence of different heat inactivated bacteria alone (control), or with either FP1 or FPl-La-C-Van. The compounds were incubated with the bacteria for 20min and then either washed 3 times (washed, concentration of compound estimated to be less than 10 nM) or loaded directly. Error bars are SEM with data from one biological donor.
Detailed description of the invention
There is increasing concern that antibiotic resistance in bacteria may render current antibiotics ineffective, for example, few treatment options remain for many strains of Staphylococcus aureus that have developed multidrug resistance. In response to the urgent need for new treatments, a novel treatment strategy has been devised by conjugating a formylated peptide and a glycopeptide antibiotic agent. The conjugates of the invention target the bacteria directly via the antibacterial action of the glycopeptide antibiotic agent, and enhance neutrophil recruitment to the site of infection by the chemoattractant actions of the formylated peptide.
In one aspect, the present invention provides a conjugate of formula (I):
Figure imgf000015_0001
or a pharmaceutically acceptable salt thereof, wherein
GPA is a glycopeptide antibiotic agent;
L is a linker moiety; and
FP is a chemotactic formylated peptide.
Reference to a “glycopeptide antibiotic agent” will be understood to mean antibiotic agents with structures containing either a glycosylated cyclic or polycyclic nonribosomal peptide, These antibiotics inhibit the cell wall structure of susceptible organisms (principally Gram- positive cocci) by inhibiting peptidoglycan synthesis. First -generation glycopeptide antibiotics include vancomycin, teicoplanin, and ramoplanin; second-generation semi- synthetic glycopeptide antibiotics include oritavancin, dalbavancin, and teiavancin, The term “glycopeptide antibiotic agent” also includes aglycone derivatives of antibiotic agents, for example, vancomycin aglycone or teicoplanin aglycone.
In one embodiment, the glycopeptide antibiotic agent is selected from vancomycin, vancomycin aglycon, vancomycin desvancosamine, desmethyl vancomycin, dalbavancin, oritavancin, teicoplanin, teiavancin, ramoplanin, decaplanin, chloroeremomycin, teicoplanin A2-2, ristocetin A, eremomycin, balhimycin, actinoidin A, complestanin, chloropeptin 1, kistamycin A, avoparcin, A40926, oritavancin and derivatives thereof. In a preferred embodiment, the glycopeptide antibiotic agent is vancomycin.
The term “linker” as herein used relates to the part of the conjugate that links the glycopeptide antibiotic agent to the chemotactic formylated peptide. It will be understood that the linker should be selected such that it does not compete with the glycopeptide antibiotic agent or the chemotactic formylated peptide. The linker group should be of a length of between 1 nm to 50 nm in order to allow the glycopeptide antibiotic agent to interact with the bacteria unhindered by the chemotactic formylated peptide. In one embodiment, the linker group will comprise one or more polyethelene glycol units. In another embodiment it is envisaged that the linker, or subunits of the linker, may be amino acid residues, derivatised or functionalised amino acid residues, polyethers, ureas, carbamates, sulphonamides or other subunits that provide adequate distance between the glycopeptide antibiotic agent and the chemotactic formylated peptide without interfering in the function of either group.
In one embodiment the linker is represented by the formula (II):
Figure imgf000016_0001
wherein
X is the attachment group between the linker and the glycopeptide antibiotic agent and is selected from -Ci-CioalkylC(O)-, -C2-CioalkenylC(0)-, -C2-CioalkynylC(0)-, -Ci- CioalkylNH-, -C2-CioalkenylNH- -C2-CioalkynylNH- -Ci-CioalkylO-, -C2-
CioalkenylO-, -C2-CioalkynylO-, -Ci-CioalkylS-, -C2-CioalkenylS-,or -C2- CioalkynylS-; or
X is an optionally C-terminal amidated amino acid wherein the amino acid is attached to the glycopeptide antibiotic agent via a side-chain functional group; m is 0, 1 or 2; n and p are independently at each occurrence 1 or 2; and denotes the point where the linker is conjugated to the formylated peptide.
In another embodiment the linker is represented by the formula (III): wherein
Figure imgf000017_0001
Y is the point of attachment between the linker and the glycopeptide antibiotic agent and is selected from -C(O)-, -NH-, -0-, or -S- m is 0, 1 or 2; n and p are independently at each occurrence 1 or 2; r is from 1 to 10; and denotes the point where the linker is conjugated to the formylated peptide.
In a further embodiment, the linker is represented by a moiety of the formula (XX):
each occurrence of R11 is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue; m is an integer from 1 to 80; and n is an integer from 0 to 1.
In other embodiments, the linker is represented by a moiety of the formula (XXI):
Figure imgf000018_0001
wherein m is an integer from 0 to 40; n is an integer from 0 to 1 ; each occurrence of o is independently an integer from 1 to 5; each occurrence of R11 is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue.
In a further embodiment, the linker is represented by a moiety of the formula (XXII):
Figure imgf000019_0001
wherein m is an integer from 0 to 40; n is an integer from 0 to 1 ; each occurrence of o is independently an integer from 1 to 5; each R12 is independently NH or O; and each occurrence of R11 is independently any side chain of a naturally occurring, derivatised or functionalised amino acid residue. The conjugates of the invention comprise a chemotactic formylated peptide conjugated to the glycopeptide antibiotic agent via the linker moiety.
Formylated peptides are one of the key chemoattractants recognised by neutrophils alongside complement component 5a (C5a) and chemokines. Formylated peptides are found solely in prokaryotic systems including mitochondria, as addition of a formyl group on the N-terminal methionine is a modification present exclusively in prokaryotic proteins. Formylated peptides are released as bacterial waste products or from damaged mitochondria, and are a signal of infection recognised in humans, i.e. pathogen-associated molecular pattern (PAMP). One source of formylated peptides are bacterial signal peptides that are cleaved off by membrane proteases that are subsequently released into the surrounding environment (Bufe, B.; et ah, J. Biol. Chem. 2015, 290 (12), 7369-7387). All N-terminal formylated methionine residues are chemotactic (Schiffmann, E.; Corcoran, B. A.; Wahl, S. M., Proc. Natl. Acad. Sci. U. S. A. 1975, 72 (3), 1059-1062). N-formyl- methionyl-leucyl-phenylalanine (fMLF), a formylated peptide isolated from the supernatant of Escherichia coli culture, was found to be the most potent stimulator of chemotaxis out of the tripeptides and is often used in standard transwell cell migration assays as a reference against which the chemotactic properties of other substances are tested (Marasco, W. A.; et al., J. Biol. Chem. 1984, 259 (9), 5430-5439). N-formyl- methionyl-isoleucyl-phenylalanyl-leucine (fMIFL), a formylated peptide produced by S. aureus, induces chemotaxis with even greater potency than fMLF (Rot, A.; et al., Proc. Natl. Acad. Sci. U. S. A. 1987, 84 (22), 7967-7971).
Formylated peptides are detected by formyl peptide receptor 1 (FPR1) and formyl peptide receptor 2 (FPR2), GPCRs present in neutrophils as well as some other cell types in humans (Fi, F.; et al., J. Leukoc. Biol. 2016, 99 (3), 425-435). FPR1 is the most important receptor of the FPR family for chemotaxis, as it binds the majority of formyl peptides with high affinity and induces chemotaxis with high potency (Bloes, D. A.; Kretschmer, D.; Peschel, A., Nat. Rev. Microbiol. 2015, 13 (2), 95-104). FPR1 shares 69% homology in amino acid sequence with FPR2, but FPR2 binds formyl peptides with much lower affinity (Dorward, D. A.; et al., Am. J. Pathol. 2015, 185 (5), 1172-1184). FPR2 has more functions and can bind a wider variety of ligands while the function of a third member of the FPR family, formyl peptide receptor 3 (FPR3), remains largely unknown. In addition to chemotaxis, formylated peptides also stimulate superoxide formation and degranulation from neutrophils as classic antibacterial defences, and hence highlight the importance of formylated peptides in activating host defence mechanisms (Fi, F.; et al., J. Leukoc. Biol. 2016, 99 (3), 425-435).
Formylated peptides are the most potent chemoattractants identified to date. The importance of formylated peptides in triggering the immune response has been established in several studies. FPRs are not crucial for healthy host function except when fighting an infection, as a deletion study showed that Fprl knockout mice have a normal healthy phenotype (Gao, J. L.; Lee, E. J.; Murphy, P. M., J. Exp. Med. 1999, 189 (4), 657-662); however, they have significantly delayed neutrophil migration and reduced superoxide production in response to Listeria infection, leading to increased mortality rates. Formylated peptides are necessary for neutrophils to mount a successful immune response as host-derived chemoattractants alone are insufficient. This was also discovered when S. aureus culture supernatant containing deformylated peptides was injected into mice triggered reduced neutrophil migration to the site of injection than the wild type culture supernatant (Di rr, M. C.; et al. Cell. Microbiol. 2006, 8 (2), 207-217). Hence formylated peptides are chemoattractants that play a major role in triggering neutrophil migration.
For the avoidance of doubt, the term “chemotactic formylated peptide” as used herein refers to a formylated peptide that acts as a chemoattractant that is recognised and results in the chemotaxis of neutrophils, i.e., the neutrophils undergo directed movement along an increasing chemical gradient. The peptides themselves do not undergo movement along an increasing chemical gradient. The formylated peptides act by binding the formyl peptide receptors of neutrophils.
In one embodiment, the formylated peptide has the sequence: f-MLFG-R5- wherein f represents a formyl moiety;
R5 is selected from NH, O or S; and one or two of the residues methionine, leucine, phenylalanine or glycine may be substituted with a naturally or non-naturally occurring amino acid. In another embodiment, one or two of the residues methionine, leucine, phenylalanine or glycine may be substituted with a naturally or non-naturally occurring amino acid as follows:
Methionine may be substituted with an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline;
Leucine may be substituted with an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine;
Phenylalanine may be substituted with an amino acid selected from tyrosine, aspartic acid, 4-fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
Glycine may be substituted with an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, proline, or phenylalanine.
In a further embodiment, one of methionine, leucine, phenylalanine or glycine is substituted with naturally or non-naturally occurring amino acid.
In another embodiment, the formylated peptide, or pharmaceutically acceptable salt thereof, has the sequence fMILF-R5-, fMIVIL-R5- or fMLP-R5-; wherein R5 is as defined above.
In a further embodiment, the formylated peptide, or pharmaceutically acceptable salt thereof, is selected from:
Figure imgf000023_0001
wherein
R1 represents a side chain of an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline; R2 represents a side chain of an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine;
R3 represents a side chain of an amino acid selected from tyrosine, aspartic acid, 4- fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
R4 represents a side chain of an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
R4a represents a side chain of an amino acid selected from leucine, arginine, glutamic acid, glutamine, histidine, serine, or phenylalanine; and
R5 is selected from NH2, OH, or SH.
The formylated peptides defined above are conjugated to the linker via the C-terminus or the derivatised C-terminus defined by R5.
In another embodiment, the formylated peptide, or pharmaceutically acceptable salt thereof, is selected from those listed in Table 1:
Table 1. Formylated peptides of the invention
Figure imgf000026_0001
Figure imgf000027_0001
As used herein, the term "alkyl", used either alone or in compound words, denotes straight chain or branched alkyl. Prefixes such as "C2-C10" are used to denote the number of carbon atoms within the alkyl group (from 2 to 10 in this case). Examples of straight chain and branched alkyl include methyl, ethyl, n -propyl, isopropyl, n-butyl, sec-butyl, /-butyl, n-pcntyl, hexyl, heptyl, 5-methylheptyl, 5-methylhexyl, octyl, nonyl, decyl, undecyl, dodecyl and docosyl (C22).
The term "alkenyl", used either alone or in compound words, denotes straight chain or branched hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or polyunsaturated alkyl groups as previously defined. Prefixes such as "C2-C20" are used to denote the number of carbon atoms within the alkenyl group (from 2 to 20 in this case). Examples of alkenyl include vinyl, allyl, 1 -methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 1-hexenyl, 3-hexenyl, 1-heptenyl, 3- heptenyl, 1-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3- butadienyl, 1,4-pentadienyl, 1,3-hexadienyl, 1,4-hexadienyl and 5-docosenyl (C22).
The term "alkynyl", used either alone or in compound words, denotes straight chain or branched hydrocarbon residues containing at least one carbon to carbon triple bond. Prefixes such as "C2-C20" are used to denote the number of carbon atoms within the alkenyl group (from 2 to 20 in this case).
As used herein, the term “optionally substituted” typically refers to where a hydrogen atom on a group has been substituted with a non-hydrogen group. Any optionally substituted group may bear one, two, three or more optional substituents.
As used herein, reference to an amino acid "side chain" takes its standard meaning in the art. Examples of side chains of amino acids are shown below:
Figure imgf000028_0001
side chain of side chain of side chain of side chain of side chain of a,g-diaminobutyric acid arginine histidine threonine cysteine
Figure imgf000028_0002
side chain of side chain of side chain of side chain of side chain of lysine ornithine glutamatic acid glutamate glutamine
Figure imgf000028_0003
side chain of side chain of side chain of side chain of side chain of aspartic acid aspartate asparagine serine leucine
Figure imgf000028_0004
side chain of side chain of methionine phenylalanine As used herein, non-naturally occurring amino acids include any compound with both amino and carboxyl functionality, derivatives thereof, or derivatives of a naturally occurring amino acid. These amino acids form part of the peptide chain through bonding via their amino and carboxyl groups. Alternatively, these derivatives may bond with other natural or non-naturally occurring amino acids to form a non-peptidyl linkage.
In addition to the negatively charged side chains shown above, it will be appreciated that a number of the side chains may also be protonated and so become positively charged, such as the side chain of lysine. The present invention contemplates within its scope these protonated side chains as well.
It will be understood that the compounds of the present invention may exist in one or more stereoisomeric forms (e.g. diastereomers). The present invention includes within its scope all of these stereoisomeric forms either isolated (in, for example, enantiomeric isolation), or in combination (including racemic mixtures and diastereomic mixtures). The present invention contemplates the use of amino acids in both L and D forms, including the use of amino acids independently selected from L and D forms, for example, where the peptide comprises two cyclohexylalanine residues, each residue may have the same, or opposite, absolute stereochemistry.
The invention thus also relates to compounds in substantially pure stereoisomeric form with respect to the asymmetric centres of the amino acid residues, e.g., greater than about 90% de, such as about 95% to 97% de, or greater than 99% de, as well as mixtures, including racemic mixtures, thereof. Such diastereomers may be prepared by asymmetric synthesis, for example, using chiral intermediates, or mixtures may be resolved by conventional methods, e.g., chromatography, or use of a resolving agent.
Additionally, the compounds of the invention are intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including the hydrated as well as the non- hydrated forms.
It is to be understood that all of the compounds of the invention described above will further include bonds between adjacent atoms and/or hydrogens as required to satisfy the valence of each atom. That is, double bonds and/or hydrogen atoms are typically added to provide the following number of total bonds to each of the following types of atoms: carbon: four bonds; nitrogen: three bonds; oxygen: two bonds; and sulfur: two, four or six bonds. It is also to be understood that definitions given to the variables of the generic formulae described herein will result in molecular structures that are in agreement with standard organic chemistry definitions and knowledge, e.g., valency rules.
General strategies for synthesising the compounds of the invention are outlined below.
Known solid or solution phase techniques may be used in the synthesis of the peptides of the present invention, such as coupling of the N- or C-terminus to a solid support (typically a resin) followed by step-wise synthesis of the linear peptide. Protecting group chemistries for the protection of amino acid residues, including side chains, are well known in the art and may be found, for example, in: Theodora W. Greene and Peter G. M. Wuts, Protecting Groups in Organic Synthesis (Third Edition, John Wiley & Sons, Inc, 1999), the entire contents of which is incorporated herein by reference.
Methods for the preparation of conjugates as described herein will be apparent to those skilled in the art and will comprise the steps of a) defining the distance between (a) the C- terminus of the formylated peptide and a binding and/or interaction site of the glycopeptide antibiotic agent; b) selecting a linker which is capable of spanning the distance as defined in (a); and c) bonding the formylated peptide and the glycopeptide antibiotic agent via the linker as selected in (b).
Corresponding working examples for such a method are given herein and are illustrated in the appended examples. The person skilled in the art is in a position to deduce relevant binding sites or interaction sites of a given or potential glycopeptide antibiotic agent and, accordingly, to determine the distance required between (a) the C-terminus of the formylated peptide and a binding and/or interaction site of the glycopeptide antibiotic agent. Such methods comprise, but are not limited to molecular modelling, in vitro and/or molecular-interaction or binding assays (e.g. yeast two or three hybrid systems, peptide spotting, overlay assays, phage display, bacterial displays, ribosome displays), atomic force microscopy as well as spectroscopic methods and X-ray crystallography. Furthermore, methods such as site-directed mutagenesis may be employed to verify deduced interaction sites of a given glycopeptide antibiotic agent or of a candidate glycopeptide antibiotic agent and its corresponding target.
The skilled addressee will understand that the selection of a linker comprises the selection of linkers known in the art as well as the generation and use of novel linkers, for example, by molecular modelling and corresponding synthesis or further methods known in the art. The term "spanning" as used herein with reference to step b) refers to the length of the linker selected to place the glycopeptide antibiotic agent at the correct locus on a bacteria while enabling the formylated peptide to function in its role as a chemoattractant.
The skilled addressee will understand that the purpose of the linker moiety is to connect the glycopeptide antibiotic agent to the formylated peptide in order to allow the glycopeptide antibiotic agent to interact with the bacteria while the formylated peptide acts as a chemoattractant. The glycopeptide antibiotic agent and the linker will contain functional groups allowing for the two to be covalently bonded. The nature of the functional group of the glycopeptide antibiotic agent is in no way limited and may include, for example, an amine group that forms an amide bond with the linker, or a hydroxyl or carboxylic acid group that forms and ether or ester bond with the linker. Strategies for coupling the formylated peptide, linker and glycopeptide antibiotic agent are known in the art, for example, as described in Jeet, K.; Ronald, T. R., Advances in Bioconjugation. Current Organic Chemistry 2010, 14 (2), 138-147; Kolmel, D. K.; Kool, E. T., Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chemical Reviews 2017, 117 (15), 10358-10376; or Zheng, M.; Zheng, L.; Zhang, P.; Li, J.; Zhang, Y. Development of Bioorthogona! Reactions and Their Applications in Bioconjugation. Molecules 2015, 20, 3190-3205
As an example, vancomycin has three potential sites for connection to the linker as illustrated below, being the primary amine on the vancomycin sugar (V-linked, i), the secondary methyl amine (N-linked, ii) and the carboxylic acid (C-linked, iii). Coupling the linker to the primary amine is straightforward and high yielding. As the primary amine is more reactive, it is required to be protected with a protecting group such as a Boc- protecting group before the linker can be coupled to secondary amine (ii). Similarly, the carboxylic acid cannot be reacted directly, as activating the acid will cause it to react with the amine groups on vancomycin. A Boc protecting strategy may be used to first mask the primary amine and then functionalise the other two positions. After protection, the methylated amine can be functionalised, for example, by coupling with 5-azidopentanoic acid and the carboxylic acid can be functionalised by coupling with 3-azido-l- propanamine, after which they may be deprotected.
Figure imgf000032_0001
The skilled addressee will also understand that selection of the functional group at the end of the linker that connects with the formylated peptide will be dictated primarily by any available functional groups on the formylated peptide of choice. As an example, if the formylated peptide comprises a free amine or carboxylic acid group, it is envisaged that the functional group of the linker will comprise a complementary carboxylic acid or amine to form an amide bond.
The conjugates and methods of the present invention may be used in the treatment and/or prevention of a range of bacterial infections. As used herein, treatment may include alleviating or ameliorating the symptoms, diseases or conditions associated with the microbial infection being treated, including reducing the severity and/or frequency of the microbial infection. As used herein, prevention may include preventing or delaying the onset of, inhibiting the progression of, or halting or reversing altogether the onset or progression of the particular symptoms, disease or condition associated with a microbial infection.
The bacterial infection may be caused by one or more species selected from one or more of the Gram-positive bacterial genera: Actinobacteria, Bacillus, Clostridium, Corynebacterium, Enterococcus, Listeria; Nocardia, Staphylococcus, and Streptococcus. Specific examples include, but are not limited to, Listeria monocytogenes and Staphylococcus aureus.
Accordingly, in embodiment the invention provides a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate according to the invention, or a pharmaceutically acceptable salt thereof.
In another embodiment the invention provides a conjugate according to the invention, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a bacterial infection in a subject in need thereof.
In a further embodiment the invention provides use of a conjugate according to the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a bacterial infection in a subject in need thereof. In one embodiment the bacterial infection is a Gram-positive bacterial infection from the genera Actinobacteria; Bacillus, Clostridium, Corynebacterium, Enterococcus, Listeria, Nocardia, Staphylococcus, or Streptococcus. In a particular embodiment the Grampositive bacterial infection is caused by methicillin-resistant Staphylococcus aureus.
In another embodiment the bacterial infection may be caused by one or more species selected from one or more of the Gram-negative bacterial genera: Acinetobacter; Actinobacillus; Bartonella', Bordetella', Brucella', Burkholderia', Campylobacter, Cyanobacteria', Enterobacter, Erwinia', Escherichia', Francisella', Helicobacter, Hemophilus', Klebsiella', Legionella', Moraxella', Morganella; Mycobacterium', Neisseria', Pasteurella', Proteus', Providencia', Pseudomonas', Salmonella', Serratia', Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia. Specific examples include, but are not limited to, infections caused by Helicobacter pylori, uropathogenic Escherichia coli, Mycobacterium tuberculosis and Pseudomonas aeruginosa.
The term “subject” is intended to include organisms such as mammals, e.g. humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g. a human suffering from, at risk of suffering from, or potentially capable of suffering from a bacterial infection. In another embodiment, the subject is a cell.
The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a conjugate as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
The term “composition” is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers. While the conjugates as hereinbefore described, or pharmaceutically acceptable salts thereof, may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the conjugate is within the scope of the invention. For example, the conjugate could be administered with one or more therapeutic agents in combination. The combination may allow for separate, sequential or simultaneous administration of the conjugate as hereinbefore described with the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition.
The term “combination”, as used herein refers to a composition or kit of parts where the combination partners as defined above can be dosed dependently or independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points. The combination partners can then be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combination can be varied, e.g., in order to cope with the needs of a patient sub -population to be treated or the needs of the single patient which different needs can be due to age, sex, body weight, etc. of the patient.
As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the conjugate care should be taken to ensure that the activity of the conjugate is not destroyed in the process and that the conjugate is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the conjugate by means known in the art, such as, for example, micro encapsulation or coating (such as the use of enteric coating). Similarly, the route of administration chosen should be such that the conjugate reaches its site of action. Those skilled in the art may readily determine appropriate formulations for the conjugates of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA. The conjugates as hereinbefore described, or pharmaceutically acceptable salt thereof, may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery. The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi.
The solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the conjugate, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolarity, for example, sugars or sodium chloride. Preferably, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.
Sterile injectable solutions are prepared by incorporating the active conjugate in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.
Other pharmaceutical forms include oral and enteral formulations of the present invention, in which the active conjugate may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard- or soft-shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active conjugate may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active conjugate in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active conjugate may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active compound to specific regions of the gut.
Liquid formulations may also be administered enterally via a stomach or oesophageal tube.
Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the conjugates of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like.
The present invention also extends to any other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions.
The compounds of the present invention may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases. The conjugates may also be administered using a nebuliser.
Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
As mentioned above the principal active ingredient may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 pg to about 200 mg. Expressed in proportions, the active compound may be present in concentrations ranging from about 0.25 pg to about 200 mg/mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
As used herein, the term "effective amount" refers to an amount of conjugate which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur once, or at intervals of minutes or hours, or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. A typical dosage is in the range of 1 pg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
The invention will now be described with reference to some specific examples and drawings. However, it is to be understood that the particularity of the following description is not to supersede the generality of the invention as hereinbefore described.
Examples
1. Peptide Synthesis - General
Peptides were synthesised on PSDRAM lanterns (37 pmol/lantem) purchased from Mimotopes (Australia), or on Rink Amide NovaPEG resin (681.8 mg, loading 0.3 mmol) purchased from Novabiochem (Germany). Fmoc -protected L-amino acids were purchased from Sigma-Aldrich (MO, USA), except for Fmoc-Met(0)-OH, Fmoc-Met(02)-OH, Fmoc-Nle-OH, Fmoc-Nva-OH, Fmoc-a-t-butyl-Gly-OH (Fmoc-t-Feu-OH), Fmoc-Phe(4- F)-OH, Fmoc-Phe(4-NH2), Fmoc-Phe(4-Cl)-OH, Fmoc-Phe(4-CN)-OH and Fmoc-NH- (PEG)2-OH, which were purchased from Merck (NJ, USA); Fmoc-Pra-OH which was purchased from Iris Biotech (Germany); and formyl-Met-OH, which was purchased from Bachem (Switzerland). Peptide chain assembly on resin was carried out on a Protein Technologies Tribute batch-wise peptide synthesizer (AZ, USA). 1.1 LC-MS
Analyses were carried out on a Shimadzu High Performance Liquid Chromatograph coupled to Mass Spectrometer LCMS-2020 (ESI, operating both in positive and negative mode) equipped with a SPD-20A Prominence Photo Diode Array Detector and a LC- 20AD solvent delivery module. Analytical separations were performed on a Waters XBridge BEH300 Prep C18 column (10 pm, 4.6 x 250 mm) (MA, USA). The solvents used were water + 0.1% formic acid (solvent A) and HPLC-grade ACN + 0.1% formic acid (solvent B).
1.2 Preparative RP-HPLC
Purifications were carried out on a Shimadzu High Performance Liquid Chromatograph equipped with a SPD-M20A Prominence Photo Diode Array Detector and two LC-20AP pumps. Preparative separations were performed on a Waters XBridge BEH300 Prep C18 column (5 pm, 19 x 150 mm) at a flow rate of 10 mL/min. The solvents used were water + 0.1% TFA (solvent A) and HPLC-grade ACN + 0.1% TFA (solvent B).
2. Synthesis of Peptide Library
Peptides were completed using a combine/split method where lanterns were mixed for common steps such as washing, amino acid coupling and deprotection steps and split for coupling specific amino acids when required. To a 20 mL glass vial, polystyrene lanterns with Fmoc-protected Rink amide linker were added (loading 37 pmol/lantern). Lanterns were swelled in DCM for 30 min. (i) Fmoc deprotection: 20% piperidine in DMF (20 min), followed by 5 x 3 min DMF washes.
(ii) Coupling conditions: Fmoc-protected amino acids (4 eq/lantem) were activated with HCTU (3.8 eq/lantem) and DIPEA (8 eq/lantem) for 5 min, and then added to the lanterns, and allowed to couple for 1 h with shaking. Following coupling, lanterns were washed in 4 x 3 min DMF washes. The same conditions were used for coupling of N-formyl methionine.
(iii) Acetylation: Lanterns were placed in AciO/DIPEA/D F (5:5:90) and allowed to react overnight with shaking.
(iv) Formylation: As previously described (Chen, F. M. F.; Benoiton, N. L., Synthesis 1979, 1979 (09), 709-710). Formic acid (6 eq) was dissolved in DCM on an ice bath with stirring. EDC (3 eq) was gradually added to the solution. 15 min after EDC was fully dissolved, DIPEA (3 eq) was added, and the lanterns were placed in the solution and allowed to react overnight with shaking.
(v) Cleavage: Lanterns were split and placed into individual 15 mL Falcon tubes. Peptides were cleaved in solution of TFA/FbO/TIPS (95:2.5:2.5) with shaking for 1 h (with exception of 2 h cleavage when the peptide contained Arg). Lanterns were removed and rinsed with TFA followed by DCM. The solution was concentrated under N2, and then precipitated in diethyl ether. The precipitate was washed 2-3 times in cold diethyl ether. The diethyl ether was evaporated in the fume hood, leaving dry crude product in -20% yield. The crude was purified by preparative RP-HPLC.
A library of 24 formylated peptides were synthesised and characterised as shown in Table 2 below.
Table 2. Library of formylated peptides
Figure imgf000042_0001
Figure imgf000043_0001
Two comparative peptides that were not formylated were also synthesised and characterised as shown below in Table 3. Table 3. Comparative peptides
Figure imgf000043_0002
3. Synthesis of peptides with alkyne functionality for click chemistry
Figure imgf000044_0001
Rink amide NovaPEG resin (Novabiochem) (681.8 mg, loading 0.3 mmol) was placed in a syringe with a filter frit and swelled in DCM for 30 min, then flushed with DMF (3 x 30 sec). Fmoc-protected propargylglycine (Pra) (2 eq) was activated with HCTU (1.95 eq) and DIPEA (4 eq) for 5 min, and then added to the resin. Coupling was completed overnight, followed by DMF washing (3 x 30 sec). The resin was then dried with diethyl ether and divided into three batches for linkers a, b and c (La, Lb and Lc). The Fmoc protecting group of the second batch Lb and third batch Lc was removed with 20% piperidine in DMF for 30 min, followed by DMF washing (4 x 30 sec). Two eq Fmoc- NH-(PEG)2-C02H (13 atoms, see below) was then activated with HCTU (1.95 eq) and DIPEA (4 eq) for 5 min and added to the resin of batch two and three.
Figure imgf000044_0002
Fmoc-NH-PEG(2)-C02H (13 atoms)
After coupling overnight, the resin was washed with DMF (3 x 30 sec). The third batch Lc was again deprotected and Fmoc-NH-(PEG)2-C02H (13 atoms) was coupled under the same conditions. The three batches of resin were dried with DCM and the FP1 sequence was completed in an automated synthesizer. The resins were then dried with DCM and cleaved in TFA-H2O-TIPS (95:2.5:2.5). The crude products, FPl-La, FPl-Lb and FP1- Lc were purified by preparative RP-HPLC, giving pure product in 15-19% yields.
4. Azide functionalisation of vancomycin for click chemistry
Figure imgf000045_0001
Scheme 1. Functionalisation of vancomycin
4.1 Boc protection of amine on vancosamine Vancomycin HC1 (1 eq) and NaHCO3 (1.5 eq) were dissolved in 1:1 water/dioxane at ~34 mM and cooled in an ice bath. Boc anhydride (1.5 eq) was added dropwise. The reaction was stirred and left overnight. The product was precipitated in acetone and washed twice in acetone, giving crude (3) in 89% yield. 4.2 Functionalisation of amine on vancosamine
5-Azidopentanoic acid (1.1 eq) was pre-activated with COMU (1.1 eq) and TEA (2 eq) in DMF for 5 min. This solution was added onto vancomycin HC1 (1 eq) dissolved in DMF at -20-50 mM, stirred overnight and analyzed by FCMS. The product was precipitated in diethyl ether and washed twice with diethyl ether, giving crude in -85-98% yield. The FC analysis allowed to determine that the ratio between 4 compounds, namely vancomycin, azidopentanoyl on the N-terminal, azidopentanoyl on the vancosamine moiety and the double addition that were all isolated by RP-HPFC preparative (30%/ 40%/ 10%/ 20%).
4.3 Functionalisation of the carboxylic acid
The C-terminal carboxylic acid of vancomycin HC1 (1 eq) was activated with COMU (1 eq) and triethylamine (TEA, 1 eq) in dimethylformamide (DMF) at -30 mM. After 5 minutes of pre-activation, either modified with 3-azidopronamine (2 eq) or propargyl amine (2 eq) were added. The reaction was stirred overnight, analyzed by FCMS (see supplementary information) after which the product was precipitated in diethyl ether and washed twice with diethyl ether, giving crude in -80% yield, that was purified by RP- HPFC preparative (see supplementary information). 4.4 Boc removal
5i OR 6i (1 eq) was dissolved in DCM at -10 mM and TFA was added at 10%, giving a sticky solution after a few minutes. After lh, the solution was concentrated by rotary evaporation and solubilised in methanol. The products were precipitated in diethyl ether and washed twice with diethyl ether, giving crude products 5 and 6 in 95-98% yield of which -50% had vancosamine cleaved (FC-MS). Formation of vancomycin-peptide conjugates by click chemistry
Figure imgf000047_0002
Figure imgf000047_0001
Scheme 2. Click reactions for conjugating formylated peptides and vancomycin
Reactions were performed in 1.5 ml Eppendorf tubes at ~3 mM. 1 eq of peptide-linker (FPl-La, FPl-Lb or FPl-Lc) was dissolved in -100 pi DMF and 1.1 eq functionalised vancomycin (2, 5 or 6) was dissolved in -500 mΐ distilled water containing 6 eq ascorbate (pH 8.4). Solutions were well mixed and dissolved. CuSO4 (3 eq) was added, leading to rapid formation of precipitate. The reaction was analysed by LC-MS after 1 h of shaking and the product was purified directly by preparative RP-HPLC, giving product with -95- 100% purity in 25-47% yields.
6. Formation of teicoplanin-peptide conjugates by click chemistry
The sugars from Teicoplanin were removed using a mixture of HC1/ isopropanol to enable the selective modification of either N-terminal amino group or the C-terminal carboxylic acid group (Scheme 3). The N-terminal azidopentyl-teicoplanin aglycone was obtained after the coupling of 5-azidopentanoic acid with DIC and Oxyma to the teicoplanin aglycone. The C-terminal azidopropyl-teicoplanin aglycone was obtained in three steps involving the protection the N-terminal amine group with a Boc group, the C-terminal modification with 3-azidopropyl amine and the removal of the Boc group with a solution of TFA/ DCM. Click chemistry between the formylated peptide (fMLFK-Pra-NH2) and the azido-funtionalised teicoplanin aglycones afforded the formylated peptide-teicoplanin aglycone conjugates bearing the formylated peptide either in N-or C-terminal.
Figure imgf000049_0001
Scheme 3. Conjugation between formylated peptide and teicoplanin aglycone. Chemical reactions: a) HC1 (12M)/ isopropanol (2:8), 80°C, 75min; b) Boc20, NaOH (1M), 1,4- dioxane, 4°C to RT, 15h; c) 3-azidopropylamine, DIC, Oxyma, DMF, RT, 15h; d) TFA/ DCM (50: 50), RT, 30min; e) 5-azidopentanoic acid, DIC, Oxyma, DMF, RT, 15h; f) fMLFK-Pra-NH2, CuS04, Sodium ascorbate, H20/ DMF (90: 10), RT, 15h.
7. Synthesis of FPR2 antagonist, RhB-PB-10 peptide
Solid phase peptide synthesis was used assemble the peptide starting from Wang resin that was acylated with Fmoc-L-Arg(Pbf)-OH (Scheme 4). Peptide elongation was performed using a solution of 20% piperidine for Fmoc removal and DIC/ Oxyma for Fmoc-amino acid or rhodamine B coupling. The completed peptide was cleaved from the resin by TFA cleavage. Scheme 4. Preparation of Rhodamine B-PB10 peptide - inhibitor of FRP2 by solid phase peptide synthesis. Chemical reactions: a) Fmoc-F-Arg(Pbf)-OH, DIC, DMAP, DMF, RT, 15h; b) acetic anhydride, DMAP, DMF, RT, 15min; c) Piperidine/ DMF (20: 80), RT, 15min; d) Fmoc-AA-OH or Rhodamine B, DIC, Oxyma, DMF, RT, lh; e) TFA/ TIS / H20 (95: 2.5: 2.5), RT, 2h.
8. Biological Testing - General
Clinical S. aureus strains (A8090 and A8094) were kindly provided by Prof Anton Peleg (Peleg, A.Y. et al. The Journal of Infectious Diseases, 2009, 199, 532-536). Powdered brain heart infusion (BHI) broth was purchased from Oxoid (UK). Powdered Mueller Hinton (MH) broth (cation adjusted) and Technical Agar were purchased from BD (MD, USA). The Easy50 magnet and Direct Human Neutrophil Isolation Kit (including Rapidspheres and Isolation Cocktail) were purchased from Stemcell Technologies (Canada). Human serum albumin (HSA), Hank’s Balanced Salt solution (HBSS), chromogenic elastase substrate N-methoxysuccinyl- Ala- Ala-Pro- Val-p-nitroanilide and Roswell Park Memorial Institute medium (RPMI) were purchased from Sigma-Aldrich. Trypan blue stain (0.4%) was purchased from ThermoFischer Scientific (MA, USA). Absorbance of bacterial cultures was read using Cary 50 Bio UV-Vis Spectrophotometer, Varian (CA, USA). Clear sterile flat-bottom 96-well polystyrene plates and black flat-bottom 96-well polystyrene plates were purchased from Greiner (Austria). Neutrophil migration was carried out in clear sterile 96-transwell plates containing 3 pm pore polycarbonate membrane, Corning (NY, USA). Breathe Easy membranes were purchased from Diversified Biotech (MA, USA).
Plates were read in a CLARIOstar microplate reader, BMG LABTECH (Australia). Absorbance readings were preceded by double orbital shaking (500 rpm, 30 s) with plates loaded into a plate stacker. Fluorescence readings were recorded at 470 nm excitation and 520 nm emission wavelength, preceded by linear shaking (500 rpm, 10 s).
8.1 Determination of number of colony forming units by absorbance
Bacteria were streak plated from glycerol stocks onto BHI agar plates and grown at 37 °C for 16 h. An overnight culture was prepared by inoculating 2-3 colonies into BHI broth at 37 °C for 16 h with shaking. The number of colony forming units (CFUs) was determined by plating 10-fold serial dilution of overnight culture onto BHI agar plates in duplicate. Absorbance at 600 nm was determined for a 1:10 dilution of overnight culture and the CFUs per unit of absorbance was calculated.
8.2 Imaging vancomycin binding to S. aureus strains 8.2.1 Fluorescent labelling with BODIPY® FL NHS Ester
Vancomycin HC1 (1 eq), fMFFK-NH2 (FP18, 1 eq) and vancomycin conjugated to FP1 via 4 PEG units and a triazolo linkage (FPl-Ld-C-Van, 1 eq) were dissolved in DMF and the pH 8 was obtained by the addition of TEA. Then, BODIPY® FL NHS Ester (l.leq) dissolved in DMF was added, the reaction was monitored by LCMS and the product was purified directly by preparative RP-HPLC. As illustrated below, BODIPY was also conjugated to vancomycin via the C terminus of the antibiotic and to a formylated peptide conjugated to vancomycin via linker a (La) to ensure the BODIPY attachment location was not altering binding profiles observed. Labelling of vancomycin was achieved following a three step protocol involving the preparation of azido-propyl vancomycin from vancomycin and BODIPY-alkyne from BODIPY® FL NHS Ester (Scheme 5). The resulting click chemistry between the BODIPY-alkyne and the azido-propyl vancomycin afforded the BODIPY labelled vancomycin.
Vancomycin. HCI
Figure imgf000052_0001
Scheme 5. C-terminal Vancomycin labelling with the fluorescent probe BODIPY. Chemical reactions: a) Propargylamine, TEA, DMF; RT, 2h; b) DIC, Oxyma, DMF, RT, lOmin; then 3-azidopropylamine, RT, 15h; c) CuS04, Sodium ascorbate, H2O/ DMF (90: 10), RT, 15h. Scheme 6. Formylated peptide labelling with the fluorescent probe BODIPY and subsequent construction of labelled fpeptide-vancomycin using click chemistry. Chemical reactions: a) BODIPY® FL NHS ester, TEA, DMF; RT, 2h; b) DIC, Oxyma, DMF, RT, lOmin; then 3-azidopropylamine, RT, 15h; c) CuS04, Sodium ascorbate, H2O: DMF (90; 10), RT, 15h. Labelled formylated peptide and vancomycin conjugates were obtained following a convergent strategy in which the formylated peptide (fMLFG-Pra-NH2) was first modified with BODIPY® FL NHS Ester (Scheme 6). Click chemistry between the fpeptide(BODIPY) and the azido-propyl vancomycin afforded the BODIPY labelled fpeptide-vancomycin conjugate.
8.2.2 STED imaging of bacteria
S. aureus strains (MSS A, ATCC 29213; MRS A, A8090; VISA, A8094) were grown to mid-exponential phase and incubated with 16 mM of either the BODIPY-fluorescein- labelled vancomycin or the FPl-Ld-C-Van conjugate labelled with BODIPY for 30 min at room temperature. The cells were collected by centrifugation and washed to remove excess unincorporated compound, followed by resuspension in fresh MHB broth. The cells were then imaged using stimulated emission depletion (STED; Abberior Instruments GmbH, Gottingen, Germany) microscope equipped with an Olympus lOOx oil objective (UPlanSApo NA = 1.4) with a 1 watt 595 nm pulsed STED laser. Image acquisition was performed with a STED laser power of 40% and 488 nm excitation laser power of 10%.
Alternatively, the cells were imaged using a Zeiss LSM 980 Airyscan 2 (Carl-Zeiss, Jena, Germany) with a 63x oil C-PlanApo 1.4NA objective using the optimal super-resolution mode. The relative binding to the septum compared to the cell wall by the BODIPY conjugates along with sialic acid binding lectic, which does not bind the septum, was performed using FUI. Three lines were drawn across the cell that intersected the cell wall and the septum. The intensity of fluorescence along these lines was plotted and the peaks determined. These peaks in fluorescence corresponded to the walls and the septum of the cell. The ratio of fluorescence at the septum compared to the wall was then calculated and plotted in GraphPad Prism. Image analysis was performed an at least 20 cells per biological replicate and at least 3 biological replicates were scored for each treatment.
8.3 Antimicrobial assay of vancomycin-peptide conjugates Compounds stocks were prepared as two-fold serial dilution at lOx the concentration required (5% DMSO in MilliQ water). Bacterial cultures of A8090 and A8094 strains were prepared by inoculating BHI broth with 2-3 colonies and growing at 37 °C for 16 h with shaking. The absorbance at 600 nm of a 1:10 dilution was determined and the culture was diluted to 5.5 x 105 CFU/ml in MH broth. Assay plates were prepared by adding 10 μL of compound stock solution in triplicate with 90 pL diluted culture to a clear 96-well plate. Vancomycin was included as positive control and 5% DMSO as negative control. Plates were covered with Breathe Easy membranes and loaded into a microplate reader kept at 35°C. Absorbance of each well was read at 595 nm every 4 h for 24 h. The percentage growth was calculated relative to the no protein control and the average of 3 biological replicated plotted using GraphPad Prism 8 software
8.4 Isolation of Human Neutrophils
Fresh human whole blood was collected in presence of EDTA under approval from Monash University Human Research Ethics Committee (project #9572) with written informed consent from healthy human donors. Neutrophils were isolated from whole blood using EasySep direct human neutrophil isolation kit (StemCell technologies) as per the manufacturer’s instructions.
To determine concentration and purity of neutrophils, a 20 pL sample was diluted with 20 pL trypan blue and cells were counted using in a haemocytometer. Cell viability was determined by trypan blue exclusion. Neutrophils were only used if purity and viability was > 98%.
8.5 Chemotaxis assay
The chemotaxis assay was performed in a 96-transwell plate with 3pm pores (Coming). The plate was prepared by adding 200 pL of peptide in chemotaxis buffer (4 mM L- glutamine, 0.5% HSA, 49% RPMI, 49% HBSS) into the bottom receiving wells at a final concentration of 1, 10, 100 and 1000 nM (0.02% DMSO). A negative control of 0.02% DMSO was used. The top of the transwell plate was loaded with 200,000 neutrophils in 75 pL chemotaxis buffer. The plate was incubated in the dark at 37 °C with 5% CO2 for
1.5 h. Neutrophils that had migrated into the bottom receiver plate were lysed by adding 10 pL/well of elastase assay buffer (500 mM Tris-HCl, 1 M NaCl, pH 7.4, and 0.5% (v/v) Triton X-100), releasing neutrophil elastase. Elastase activity was used as an indicator of neutrophil numbers, and was measured by adding the chromogenic elastase substrate N- methoxysuccinyl- Ala- Ala-Pro- Val-p-nitroanilide (Sigma) at final concentration of 1 mM (Corriden, R. el al. Journal of Biological Chemistry, 2008, 283, 28480-28486). After 30 min at room temperature, the absorbance at 405 nm was determined using a microplate reader (Clariostar plate reader, BMG). Absorbance readings were converted to number of neutrophils using a standard curve of known neutrophil numbers. All peptides were tested in duplicate with 3-4 biological replicates, ensuring different donors each time.
Human neutrophils displayed a variability of response to formylated peptides between individual donors. This variability was taken into account by determining the percentage of neutrophil recruitment relative to the recruitment observed to the fMLFG peptide at 100 nM from the same neutrophil donor. To compare data between donors, the positive control of 100 nM fMLFG (FP1) was set as 100% and all other conditions calculated relative to this. Data was plotted using GraphPad Prism 8 software.
8.6 Determining effect of conjugates on the interaction of Neutrophils with S. aureus
8.6.1 Preparation of microfludics devices
PDMS (polydimethylsiloxane, Fischer Scientific, Fair Lawn, NJ) microfluidic devices were generated through soft lithography from silicon wafers fabricated from chrome masks designed previously (Ellett, F. el al. Lab on a Chip, 2019, 19, 1205-1216). Fabrication and use of this microfluidic device for monitoring neutrophil migration and killing of live S. aureus (SH1000-GFP) or pHrodo Red S. aureus BioParticles® (Life Technologies) in cell culture medium (IMDM + 10% FBS) has been outlined in detail by the designers (Ellett el al. 2019).
8.6.2 Microfludic data acquisition and analysis
Monitoring the kinetics of neutrophil migration and phagocytosis was imaged using an automated Nikon Eclipse TiE inverted wide field microscope with stage control and perfect focus system. The biochamber maintained temperature at 37 °C and CO2 at 5% for the 8h of the experiment, where images were captured every 5 min for each field of view (FOV), with at least 4 FOV captured per experimental condition. For each experimental condition at least 12 chambers were tracked for neutrophil migration using the FIJI (Schindelin, J. et al. Nature Methods 2012, 9, 676-682) plugin Trackmate (Tinevez, J.-Y. et al. Methods, 2017, 115, 80-90). The percentage of neutrophils recruited into the chamber was determined relative to the total number of neutrophils in each FOV divided by the number of chambers visible. Data was plotted using GraphPad Prism 8 software.
8.6.3 Competition binding assay for FPR2
The fluorescently labelled FPR2 antagonist, RhB-PBlO, was used to determine the interaction of the formylated peptides with FPR2 of human neutrophils. RhB-PBlO (15uM) was preincubated with human neutrophils (5x10Λ6neutrophils/mL) in Bis-Tris buffer pH6 for lh, washed twice in Bis-Tris buffer pH6, before incubating with 8uM of peptide for 5min. Neutrophils were washed, and then resuspended in Bis Tris buffer. Rhodamine B levels of the samples were determined using microplate reader (Clariostar plate reader, BMG) with ex 550 nm, em 585 nm.
8.7 Mouse pneumonia model
Eight-week-old female Balb/c mice were anaesthetised with isoflurane, before intranasal inhalation of 107cfu S. aureus, in 50 μL of PBS. One-hour post infection (hpi) mice were treated intranasally with 50 pL of vancomycin (0.2 mg/50 pL) or equivalent molar amount of Fmlfg (FP1), FP9-La-C-Van or vehicle control (5% DMSO in PBS). Mice were humanely euthanized by CO2 inhalation at 12 hpi and lung bacterial density was assessed from four mice per treatment group. Lungs were homogenised and underwent serial dilution and plating onto solid media for CFU enumeration. Significance was determined using a Mann-Whitney test with p<0.02 being significant and calculated using GraphPad Prism 8 software.
For pathology assessment, excised lungs were fixed in 10% buffered formalin before undergoing routine histological processing (Monash University Histology Platform). Lungs were embedded in paraffin, cut into 5 pm thick sections and stained with hematoxylin and eosin. Tissue pathology was assessed by a veterinary pathologist (Dr Mark Williamson, Gribbles Pathology, Clayton, Australia). All experiments were performed in accordance with the Animal Research Ethics Committee at Monash University (MARP/2018/012). The loss of alveoli structure was quantified in the H&E sections by determining the area of white space in six FOV (1.8x1.2mm) in the outer lobes of the lung. In addition, the number of nuclei in these FOV were also determined. This was performed by thresholding the image (0-130) and then using the analyze particle function (particles 80-4000, circularity 0.05-1) in FIJI (Schindelin, J. et al. Nature Methods 2012, 9, 676-682) to count the number of nuclei detected. Data was plotted using GraphPad Prism 8 software.
9. Antibacterial activity of formylated peptide-vancomycin conjugates
The formylated peptide-vancomycin conjugates were tested for antibacterial activity against S. aureus. Two strains were used: A8090, a vancomycin-sensitive S. aureus clinical isolate, and A8094, a vancomycin-intermediate S. aureus isolate from the same patient. The inhibition of bacterial growth was tested over a range of concentrations of compound using a broth microdilution plate assay. The amount of bacterial growth is reported as a percentage of bacterial growth in absence of compound (negative control). The inhibition is then compared to vancomycin to characterise the effect of conjugation and linker length on the antibacterial activity of the conjugated vancomycin. The experiment was performed in three biological replicates, each time in triplicate.
From the inhibition curves, the IC50 (concentration of 50% growth inhibition) and minimum inhibitory concentration (MIC), i.e. concentration of 80% growth inhibition, were calculated using linear interpolation (Table 3).
Table 3. IC50 and MIC values for FPl-vancomycin conjugates
MRSA VISA
Figure imgf000058_0001
La 1.4 + 0.09 1.8 + 0.04 14 + 5 NA
Lb 2.7 + 0.1 3.7 + 0.2 NA NA
Lc 1.7 14.4
Figure imgf000059_0001
NA NA
Y-linkcd
Figure imgf000059_0002
Figure imgf000059_0003
La 6.1+ 1.5 14.5 + 1.2 23+ 0.6 30 + 6
Lb 8.2 + 1.2 15.4 + 1.2 NA NA
Lc 12.7 + 1.8 16.8 + 3.7 NT NT
Values are represented as mean ± standard deviation. NA = not active < 30 mM. NT = not tested
9.1 Activity of formyl peptide-vancomycin conjugates against vancomycin- susceptible S. aureus Vancomycin had an MIC of 0.65 mM against MRSA, the vancomycin- susceptible strain. All three of the vancomycin analogues conjugated via secondary methylated amine (Compounds FPl-La-N-Van, FPl-Lb-N-Van, and FPl-Lc-N-Van) were completely inactive against both strains of S. aureus up to 30 mM (Table 3, Figure 1). In contrast, the three vancomycin analogues conjugated via the carboxyl group (Compounds FPl-La-C- Van, FPl-Lb-C-Van and FPl-Lc-C-Van) were active against MRSA, the vancomycin- susceptible strain, in a dose-dependent manner (Figure 1, Table 3); the longer the linker, the higher the dose required to inhibit S. aureus growth.
Conjugating the formylated peptide to the methylated amine on vancomycin through any PEG linker tested completely nullified its antibacterial activity. This conjugation site maybe affecting the binding of vancomycin to the bacterial cell wall target. The antibiotic action of vancomycin is mainly attributed to its ability to bind tightly via its peptide backbone to the D-Ala-D-Ala motif of the peptidoglycan cell wall of bacteria (Barna, J. C.; Williams, D. H., Annu. Rev. Microbiol. 1984, 38, 339-357). Attachment through the methylated amine on vancomycin may be directly disrupting this binding. It can be concluded that conjugation of formyl peptide at this site is not a viable strategy for direct killing of S. aureus. As well as not being able to kill S. aureus, these compounds by extension are also likely to have much lower affinity for the bacterial cell wall and thus may not be capable of generating the chemoattractant gradient necessary for recruitment of neutrophils. The peptide backbone of vancomycin should not be affected by modification of the sugar moiety. However, a reduction in antimicrobial activity was observed by linking to through the vancosamine primary amine. This reduction may be attributed to the linkage affecting the dimerization that occurs through these sugars and is important for activity. The sugars present on vancomycin and other glycopeptide antibiotics are essential to their antibacterial activity (Nagarajan, R., Antimicrob. Agents Chemother. 1991, 35 (4), 605-609). Vancomycin is produced by Amycolatopsis orientalis, and logically, the metabolic expense of the biosynthesis of the sugars should be justified by serving an important function that provides the bacterium with a survival advantage. NMR evidence has demonstrated that the sugars on vancomycin are necessary for dimerization to occur (Gerhard, U.; et ah, J. Am. Chem. Soc. 1993, 115 (1), 232-237). It is not entirely clear how dimerization aids the antibacterial activity of vancomycin, but the ability of vancomycin to dimerise is enhanced by binding to D-Ala-D-Ala (McPhail, D.; Cooper, A., J. Chem. Soc., Faraday Trans. 1997, 93 (13), 2283-2289). Indeed, Silverman et al. (2017) demonstrated that covalently linked dimers of vancomycin showed improved activity over vancomycin itself, giving further evidence that the improvement of antibacterial activity is truly due to dimerization (Silverman, S. M.; Moses, J. E.; Sharpless, K. B., Chem. - Eur. J. 2017, 23 (1), 79-83). Although never proven in vivo , together the evidence suggests that vancomycin exhibits cooperative binding, i.e. vancomycin dimerization is enhanced when bound to the cell wall, and its binding to the cell wall is enhanced by dimerization (Williams, D. H.; Maguire, A. J.; Tsuzuki, W.; Westwell, Science 1998, 280 (5364), 711). From the experiments, it is evident that conjugation of formyl peptide to the primary amine has blocked the sugar moiety, inhibiting the dimerization of vancomycin and therefore reducing its antibacterial activity.
The three conjugates linked via the carboxyl group FPl-La-C-Van, FPl-Lb-C-Van and FPl-Lc-C-Van, were all active in killing S. aureus, although their antibacterial activity is reduced compared to vancomycin (Figure 1). Of the three, the conjugate with the shortest linker La showed the most potent antibacterial activity with an MIC of 1.85 mM, compared to vancomycin with an MIC of 0.65 pM against the same strain. The reduction in activity is not ideal; nevertheless, the conjugation of formyl peptide via the carboxyl group successfully yields compounds with retained antibacterial activity.
9.2 Activity of formylated peptide-vancomycin conjugates against vancomycin- intermediate S. aureus
The formylated peptide-vancomycin conjugates were tested against a vancomycin- intermediate strain isolated from the same patient (Figure 1). This S. aureus strain developed intermediate-level resistance from exposure to vancomycin treatment, increasing the MIC of vancomycin to 10 mM. The three vancomycin analogues conjugated via methylated amine (N-linked) that had no activity against MRS A, continued to have no activity against VISA (Figure 1). Of the three vancomycin analogues conjugated via the carboxyl group, the conjugate with the shortest linker La showed some activity against VISA; the other two analogues FPl-Lb-C-Van and FPl-Lc-C-Van, with increasing repeats of PEG did not inhibit growth at any concentration up to 30 pM (Figure 1, Table 2).
Against this strain, the V-linked conjugates with linker La inhibited bacterial growth, while the conjugate with linker Lb resulted in growth inhibition but not complete inhibition at the maximum concentration tested.
Vancomycin is clinically not effective against VISA, which by definition are strains with MIC > 16 pg/ml (11 pM); thus, conjugates were only tested up to 30 pM. At high concentrations, vancomycin is ineffective at treating VISA and VRSA because of limited membrane penetration (Rybak, M. J., Clin. Infect. Dis. 2006, 42 (Supplement_l), S35- S39). An additional issue with using high concentrations of vancomycin is its potential for nephrotoxicity and ototoxicity, especially when used in combination with other drugs (Rybak, M.; et al, Am. J. Health Syst. Pharm. 2008, 66 (1), 82). In general, alternative treatments are implemented for patients infected with S. aureus isolates that have MIC > 2 pg/ml (1.38 pM). Therefore, these results show that the antibacterial activity alone of the conjugated compounds will not achieve positive clinical outcomes for treating MRSA or VISA. However, the most critical feature of these compounds is their ability to recruit neutrophils to eliminate S. aureus. Generating the chemical gradient that leads the neutrophils to S. aureus is dependent on the ability of the compounds to bind to the cell wall. As these compounds have antibacterial activity against vancomycin-susceptible S. aureus (VSSA), they should be capable of binding to the cell wall in a similar fashion to vancomycin. This binding was confirmed by STED microscopy using Bodipy labelled versions of vancomycin alone or vancomycin with C-terminal conjugated formylated peptide FP1 with 4 PEG repeat linker (FPl-Ld-C-Van). Binding of the conjugate occurred with a similar distribution across all three S. aureus strains tested: MSSA, MRSA and VISA (Figure 3). Additionally, binding of formylated peptide linked to vancomycin with BODIPY attached through the C-terminus also displayed similar binding patterns to C- terminal labelled vancomycin across all three S. aureus strains by AiryScan confocal microscopy (Figure 4)
10. Chemotactic activity of formylated peptide-vancomycin conjugates
Given the location of the vancomycin binding site in the cell wall it was important to consider the display of formylated peptide on the surface of S. aureus. The flexible and polar poly ethylene glycol (PEG) spacer of defined lengths (0, 3, or 6 repeats) was used to select the optimum linker. Using a transwell assay to monitor neutrophil chemotaxis it was determined that attachment site on the vancomycin and length of the linker did not considerably affect neutrophil recruitment by the formylated peptide. The peak of neutrophil recruitment was observed at 100 nM for all attachment sites and linker lengths, similar to the free fMLFG (FP1, Figure 2). In addition, a decrease in neutrophil recruitment was observed at 10 nM for all three attachment sites and linker lengths compared to the FP1 alone.
Conjugating FP1 has a slight detrimental effect to its chemotactic activity. Regardless, the compounds were still potent chemoattractants with similar activity to the parent peptide. Ignoring the effect of linker size, comparing conjugation at the three different sites: the vancosamine primary amine; methylated amine; or the carboxyl group, there is little difference in chemotaxis profiles. Thus, the reduction in chemotactic activity of the conjugates compared with fMLFG is merely a consequence of conjugation, and is not a result of the particular position of attachment of formyl peptide to vancomycin. It appears that the ability of the formyl peptide moiety to stimulate chemotaxis is much the same no matter where it is conjugated onto vancomycin.
11. The effect of the sequence of the formylated peptide on neutrophil recruitment
To find the optimum formylated peptide to use as a payload a library was designed based upon the fMLFG (FP1) sequence by combinatorial peptide synthesis on lanterns (Figure 4). Each lantern was tagged and common synthetic steps could be achieved following a split/mix to speed up the elaboration of the library. Each of the residues of this sequence were modified to a variety of different natural and non-natural amino acids (Figure 4). This gave a library of formylated peptide covering a range of different hydrophobic and size profiles (Figure 4 and Table 1). Using a transwell migration assay, the chemotaxis of neutrophils to these peptides was examined at different concentrations (1-1000 nM).
It was observed that the library could be classified into 6 different profiles, depending on the concentrations responsible for the maximum recruitment of neutrophils (Figure 4 B-F). Peptides that did not contain the formyl group did not recruit neutrophils across the concentration range tested (Figure 6). No correlation could be determined between the chemotaxis profile observed and the position of the change, the solvent accessible surface area or the hydrophobicity of the formylated peptide. This was despite of the wide range of hydrophobic profiles tested (ranging from 2.5 negative to 2.5 positive compared to the original fMLFG sequence), and changes in the surface accessible area (Figure 8).
It was important that overstimulation of the immune response was avoided as can occur with potent formylated peptide sequences. As an example, inhalation of fMLF, one of the most potent of the formylated peptide sequences known, can cause rapid neutropenia and bronchial inflammation in humans (Peters, M.J., et al. Thorax 1992, 47, 284-7). The generation of the formylated peptide library based on the fMLFG sequence provided a resource in regards to diversity of sequences that contained not only non-proteolytic amino acids but also peptides with different chemotaxis potency; by having different concentrations required to induce peak neutrophil chemotaxis.
12. The effect of the sequence of the formylated peptide attachment to vancomycin on neutrophil recruitment
The diversity of peptide profiles created allowed for tailoring the formylated peptide attached to vancomycin not only in terms of hydrophobicity, stability, and size but also in regards to the strength of neutrophil recruitment. Therefore, there was a need to investigate if attachment of these peptides to vancomycin altered the neutrophil chemotaxis profile. Following the same strategy described above (formylated peptides syntheses by SPPS followed by click chemistry conjugation), representatives of each of the 6 chemotactic profiles were linked onto vancomycin through the carboxyl group linkage site via click chemistry, and neutrophil recruitment was examined. The attachment of the formylated peptide resulted in a loss of activity at lower concentrations with a shift to activity at higher concentrations, as observed with conjugation of the representative fMLFG (FP1). This change in profile to peaks 100 or 1000 nM occurred irrespective of the initial free peptide profile (Figure 6). It is important to note that vancomycin alone did not recruit neutrophils at the concentrations tested, nor did vancomycin linked to MLFG (Figure 7).
The peptides fMLFL (FP16, Figure 3D) and fMLYG (FP10, Figure 3F) that recruited low numbers of neutrophils across the concentration range, also did so when conjugated to vancomycin. These sequences were chosen for conjugation as they resulted in low neutrophil recruitment. Making them ideal to test further to see if they improve bacterial killing without overstimulating neutrophils, which can lead to deleterious effects (Berend, N., Armour, C.L. & Black, J.L. Agents Actions, 1986, 17, 466-71; Peters, M.J., et al., Thorax 1992, 47, 284-7). Using the microbroth dilution assay it was also determined that the sequence of the formylated peptide attached to vancomycin did not have an effect on antimicrobial activity. A two-fold serial dilution of the conjugates was tested from 30 to 0.46 uM against three strains of S. aureus- MSS A, MRS A, and VISA. Irrespective of the formylated peptide attached to vancomycin, the MIC against the strains of S. aureus tested remained the same (Table 4).
Table 4. MIC values for formylated peptide-vancomycin conjugates
Figure imgf000065_0001
Testing the neutrophil recruitment and direct antimicrobial killing separately allowed for the independent observation of the individual activities of the two-component drug. However, an understanding as to whether linking these two activities enhanced the neutrophil killing of S. aureus was needed. 13. Determining the effect of formylated peptide sequence on neutrophil recruitment and phagocytosis of S. aureus
An infection-on-a-chip microfluidic device was used to examine the effect of the conjugates on the kinetics of neutrophil recruitment and phagocytosis of S. aureus (Figure 9A). This technology allowed for the observation of the influence of the drugs on the host- pathogen interactions at single cell detail in real time. This was achieved by observing the migration of neutrophils into infected microchambers that contained the compounds and S. aureus (Figure 9). Imaging at 5 min intervals allowed for the observation of neutrophils phagocytosing S. aureus, as the bacteria were labelled with the pH sensitive dye, pHrodo, that becomes fluorescent in the acidic environment of the neutrophil phagosome (Figure
9).
Using this microfludic method, it was observed that the kinetics of neutrophil chemotaxis varied with the sequence of the formylated peptide and the conjugation state. The kinetics of neutrophil recruitment was slightly faster for conjugated FP1 compared to the free peptide (Figure 10). In contrast, the FP9 had similar kinetic profile for both the free and conjugated peptides (Figure 10). Increased recruitment of neutrophils into the chamber by the conjugate or free peptides also resulted in enhanced control of S. aureus growth compared to vancomycin alone. Neutrophils were recruited into the microchamber from the start of the assay. However, there was an hour delay before the fluorescence from the pHrodo dye increased above background (Figure 10).
For the FP1 peptides, despite the faster recruitment of neutrophils observed for the conjugate, there was only a slight increase in the pHrodo kinetics over the free formylated peptide. In contrast, FP9 free and conjugated had similar levels of neutrophils recruited into the microchamber, but the conjugate resulted in a quicker increase in pHrodo fluorescence, and had an increase in the amount of pHrodo fluorescence per neutrophil (Figure 7 E). This suggested that the FP9 conjugate resulted in an increase in the number of bacteria phagocytosed per neutrophil. This enhancement with the FP9-Van conjugates was observed with all donors to different extents. This is in contrast to the FP1 peptide, where similar pHrodo fluorescence per neutrophil was observed for both the free and conjugated peptide for all donors (Figure 10). However, other conjugated peptides tested of FP16 and FP10, did not display this enhanced profile across all donors (Figure 10).
This difference between formylated peptides in enhancement of phagocytosis may be due to different activation of the two main formylated peptide receptors (FPR) on neutrophils, FPR1 & FPR2. To examine this, the rhodamine B labelled FPR2 antagonist, RhB-PBlO, was added to human neutrophils and determined if the formylated peptides could compete off this antagonist (Figure 11). The fMIVIL peptide is described in literature as a FPR2 binding formylated peptide, which was confirmed in this assay as it resulted in the reduction in RhB-PBlO fluorescence in human neutrophils. In contrast, fMLF is described as a preferential activator of FPR1, and resulted in similar levels of RhB-PBlO fluorescence bound to human neutrophils as the no peptide control. FPR binding of the formylated peptide was altered by conjugating to vancomycin. The FP9-Van conjugate resulted in no loss of fluorescence suggesting it was not able to compete with RhB-PBlO binding to FPR2. This is in contrast to the FP9 peptide, which did reduce fluorescence and compete with RhB-PBlO binding.
14. Antibacterial activity of formylated peptide-teicoplanin conjugates
Attachment of the formylated peptide to the N terminus of teicoplanin aglycone, resulted in a loss in antimicrobial activity against S. aureus. While C terminal attachment of the formylated peptide had similar activity at the concentrations tested. Chemotaxis of human neutrophils to the conjugates was determined by the transwell assay. Both N and C terminal attachment resulted in recruitment of human neutrophils (Figure 12). This demonstrates that swapping the targeting element from vancomycin to other antibacterial agents such as teicoplanin aglycone is possible while maintaining both antimicrobial and chemo tactic activity.
15. Conjugate reduces inflammation and bacterial load in S. aureus mouse pneumonia
The most promising conjugate, FP9-La-C-Van was tested in a mouse pneumonia model (Figure 13). This models pneumonia, a common and severe S. aureus infection often seen in hospitalised patients. To establish MRSA pneumonia, 8-week old mice were infected intranasally with 107 CFU S. aureus. Treatment was administered 1 hpi intranasally with dose of 0.2 mg/mouse equivalent of vancomycin. This dose is 5-fold less the standard 100 mg/kg equivalent of the clinical dose of vancomycin in humans (1 mg), in regards to the area under the concentration-time curve (AUC) of total vancomycin in serum. This treatment resulted in a 2-fold reduction in MRSA loads in the lungs of FP9-La-C-Van treated mice compared to the vancomycin alone (Figure 13). Histopathology revealed that the lung tissue of the vehicle control mice had the most severe and widespread inflammation and bacterial load, resulting in loss of alveoli structure (Figure 14). The inflammation was centered on the alveoli, where there were large numbers of neutrophils, alveolar macrophages and both extracellular and intracellular bacteria. The extent of the inflammation and infection of the lung was measured by the area of alveoli air space, which was dramatically reduced in untreated infected lung tissue (Figure 15). Vancomycin treatment reduced the severity of the inflammation and bacterial numbers. The formylated peptide alone FP1 and the conjugate FP9-La-C-Van both reduced severity of the inflammation and bacterial numbers compared to the vancomycin treatment, resulting in the inflammation not extending into the lumen of bronchioles and bronchi (Figure 14).
16. Conjugate recruits neutrophils to Gram-negative bacteria
Airy scan imaging was used to determine binding location of the C terminal BODIPY labelled vancomycin and formylated peptide linked C terminal to vancomycin with a variety of heat killed bacteria (Figure 16). It was confirmed that compounds bind to the heat killed strains of MSS A, MRS A and VISA in the same pattern of binding as the live strains previously tested. No binding of the BODIPY labelled formylated peptide alone was observed to all of the heat killed bacteria tested. Both the BODIPY labelled conjugate and vancomycin were observed bound to the Gram-positive heat killed strains of Bacillus subtillus, and Listeria monocytogenes (Figure 16). Additionally the Gram-negative heat killed E.coli 0111:B4 was also bound by the BODIPY labelled compounds along with an avirulent strain of Mycobacterium tuberculosis H37 Ra (Invivogen) although to a lesser extent than the Gram-positive strains.
Neutrophil recruitment was observed for heat inactivated Gram-negative bacteria of E. coli, and Pseudomonas along with Mycobacterium in the presence of the FPl-La-C- Van. Briefly, heat killed bacteria of E.coli 0111:B4, Pseudomonas aeruginosa and avirulent strain of Mycobacterium tuberculosis H37 Ra (Invivogen) were labelled with pHrodo (Thermo). Beat killed bacteria (8x 10Λ 7 bioparticles/mL) were incubated with 1000 nM of fMLFG, FPl-La-C-Van or 1% DMSO control for 20 min at room temperature. Particles were either used directly or washed twice with 2 volumes of PBS before respending in IMDM + 10% PBS. Samples were loaded into microfluidic devices and standard neutrophil migration assay performed. As can be seen from Figure 17, bacteria incubated with FP1 and washed have less recruitment compared to FPl-La-C-Van washed. For E. coli, Pseudomonas, and Mycobacterium the recruitment of washed FPl-La-C-Van is similar to the recruitment observed for unwashed FPl-La-C-Van.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A conjugate of formula (I):
Figure imgf000070_0001
or a pharmaceutically acceptable salt thereof, wherein
GPA is a glycopeptide antibiotic agent; L is a linker moiety; and
FP is a chemotactic formylated peptide.
2. The conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the formylated peptide has the sequence: f-MLFG-NH wherein f represents a formyl moiety; and one or two of the residues methionine, leucine, phenylalanine or glycine may be substituted with a naturally or non-naturally occurring amino acid.
3. A conjugate or pharmaceutically acceptable salt thereof according to claim 2, wherein: methionine may be substituted with an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline; leucine may be substituted with an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine; phenylalanine may be substituted with an amino acid selected from tyrosine, aspartic acid, 4-fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and glycine may be substituted with an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, proline, or phenylalanine.
4. The conjugate or pharmaceutically acceptable salt thereof according to claim 2 or
3, wherein one of methionine, leucine, phenylalanine or glycine is substituted with naturally or non-naturally occurring amino acid.
5. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein L is a linker represented by the formula (II):
Figure imgf000071_0001
wherein
X is the attachment group between the linker and the glycopeptide antibiotic agent and selected from -Ci-CioalkylC(O)-, -C2-CioalkenylC(0)-, -C2-CioalkynylC(0)-, -Ci- CioalkylNH-, -C2-CioalkenylNH- -C2-CioalkynylNH- -Ci-CioalkylO-, -C2-
CioalkenylO-, -C2-CioalkynylO-, -Ci-CioalkylS-, -C2-CioalkenylS-, or -C2-
CioalkynylS-; or X is an optionally C-terminal amidated amino acid wherein the amino acid is attached to the glycopeptide antibiotic agent via a side-chain functional group; m is 0, 1 or 2; n and p are independently at each occurrence 1 or 2; and denotes the point where the linker is conjugated to the formylated peptide.
Figure imgf000072_0002
6. The conjugate or pharmaceutically acceptable salt thereof according to claim 5 wherein L is represented by the formula (III): wherein
Figure imgf000072_0001
Y is the point of attachment between the linker and the glycopeptide antibiotic agent selected from -C(O)-, -NH-, -0-, or -S- m is 0, 1 or 2; n and p are independently at each occurrence 1 or 2; r is from 1 to 10; and denotes the point where the linker is conjugated to the formylated peptide, according to claim
7. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the glycopeptide antibiotic agent is selected from vancomycin, vancomycin aglycon, vancomycin desvancosamine, desmethyl vancomycin, dalbavancin, oritavancin, teicoplanin, telavancin, ramoplanin, decaplanin, chloroeremomycin, teicoplanin A2-2, ristocetin A, eremomycin, balhimycin, actinoidin A, complestanin, chloropeptin 1, kistamycin A, avoparcin, A40926, oritavancin and derivatives thereof.
8. The conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the glycopeptide antibiotic agent is vancomycin.
9. A formylated peptide, or pharmaceutically acceptable salt thereof, selected from:
Figure imgf000073_0001
wherein
R1 represents a side chain of an amino acid selected from methionine sulfoxide, methionine sulphone, norleucine and norvaline; R2 represents a side chain of an amino acid selected from norleucine, norvaline, tert- leucine and cyclohexylalanine;
R3 represents a side chain of an amino acid selected from tyrosine, aspartic acid, 4- fluorophenylalanine, 4-chlorophenylalanine, 4-aminophenylalanine and 4- cyanophenylalanine; and
R4 represents a side chain of an amino acid selected from leucine, arginine, lysine, glutamic acid, glutamine, histidine, serine, or phenylalanine;
R4a represents a side chain of an amino acid selected from leucine, arginine, glutamic acid, glutamine, histidine, serine, or phenylalanine; and
R5 is selected from NH2, OH, or SH.
10. A pharmaceutical composition comprising a therapeutically effective amount of a conjugate according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.
11. A method of treating or preventing a bacterial infection comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.
12. The method according to claim 11, wherein the bacterial infection is a Grampositive bacterial infection from the genera Actinobacteria; Bacillus , Clostridium, Corynebacterium, Enterococcus, Listeria, Nocardia, Staphylococcus, or Streptococcus.
13. The method according to claim 12, wherein the Gram-positive bacterial infection is caused by Staphylococcus aureus.
14. The method according to claim 13 wherein the Gram-positive bacterial infection is caused by methicillin-resistant Staphylococcus aureus.
15. The method according to claim 11, wherein the bacterial infection is a Gramnegative bacterial infection from the genera Acinetobacter; Actinobacillus; Bartonella·, Bordetella', Brucella', Burkholderia', Campylobacter, Cyanobacteria', Enterobacter, Erwinia', Escherichia', Francisella', Helicobacter, Hemophilus', Klebsiella', Legionella', Moraxella', Morganella; Mycobacterium', Neisseria', Pasteurella', Proteus', Providencia', Pseudomonas', Salmonella', Serratia', Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia.
16. A conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a bacterial infection in a subject in need thereof.
17. The conjugate for use according to claim 16, wherein the bacterial infection is a Gram-positive bacterial infection from the genera Actinobacteria; Bacillus , Clostridium, Corynebacterium, Enterococcus, Listeria, Nocardia, Staphylococcus, or Streptococcus.
18. The conjugate for use according to claim 17, wherein the Gram-positive bacterial infection is caused by Staphylococcus aureus.
19. The conjugate for use according to claim 18 wherein the Gram-positive bacterial infection is caused by methicillin-resistant Staphylococcus aureus.
20. The conjugate for use according to claim 16, wherein the bacterial infection is a Gram- negative bacterial infection from the genera Acinetobacter; Actinobacillus; Bartonella', Bordetella', Brucella', Burkholderia', Campylobacter, Cyanobacteria', Enterobacter, Erwinia', Escherichia', Francisella', Helicobacter, Hemophilus', Klebsiella', Legionella', Moraxella', Morganella; Mycobacterium', Neisseria', Pasteurella', Proteus', Providencia', Pseudomonas', Salmonella', Serratia', Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia.
21. Use of a conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a bacterial infection in a subject in need thereof.
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Citations (4)

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