WO2015022504A2 - Peptide conjugates - Google Patents

Peptide conjugates Download PDF

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Publication number
WO2015022504A2
WO2015022504A2 PCT/GB2014/052439 GB2014052439W WO2015022504A2 WO 2015022504 A2 WO2015022504 A2 WO 2015022504A2 GB 2014052439 W GB2014052439 W GB 2014052439W WO 2015022504 A2 WO2015022504 A2 WO 2015022504A2
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Prior art keywords
peptide
cargo
conjugates
molecule
construct
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PCT/GB2014/052439
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French (fr)
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WO2015022504A3 (en
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Michael John Gait
Peter Joseph DEUSS
Elizabeth O'DONOVAN
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • the present invention relates to peptide conjugates.
  • CPPs cell-penetrating peptides
  • PTD protein transduction domains
  • CPPs can either be complexed with or conjugated covalently to the cargo.
  • CPP conjugates of charge neutral antisense phosphorodiamidate morpholino oligonucleotides (PMO) or peptide nucleic acids (PNA) have been shown to be very effective in induction of splicing redirection or exon skipping in cells and in vivo?
  • PMO charge neutral antisense phosphorodiamidate morpholino oligonucleotides
  • PNA peptide nucleic acids
  • the inventors have now provided a method for the rapid synthesis, and isolation of peptide-cargo conjugates in which a peptide is chemically conjugated to a cargo molecule. This advance in the art makes possible the rapid and high throughput screening of peptide-cargo conjugates for the first time.
  • a method of forming a peptide-cargo conjugate comprising:
  • the method may optionally comprise a washing step after step (i) and/or step (ii).
  • a method of forming peptide-cargo conjugates comprising:
  • the cargo molecule also comprising a tag
  • the method may optionally comprise a washing step after step (c) and/or step (d).
  • each of the plurality of cargo constructs in (b) have the same cargo molecule, and/or the same functional group and/or tag.
  • a method of forming a peptide-cargo conjugate, the conjugate comprising a peptide bonded to a cargo molecule comprising:
  • a method of forming peptide-cargo conjugates, the conjugates each comprising a peptide bonded to a cargo molecule, comprising:
  • the method may optionally comprise a washing step after step (c) and/or step (d).
  • each of the plurality of cargo constructs in (b) have the same cargo molecule, and/or the same functional group and/or tag.
  • a method of generating a library of peptide- cargo conjugates comprising:
  • the method may optionally comprise a washing step after step (c) and/or step (d).
  • a method of screening a plurality of peptide-cargo conjugates for a functional property of the conjugate comprising simultaneously assaying a plurality of different peptide-cargo conjugates for said functional property.
  • a method of generating a library of peptide- cargo conjugates, the library having a plurality of different peptide-cargo conjugates comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
  • the method may optionally comprise a washing step after step (c) and/or step (d).
  • a method of screening a plurality of peptide-cargo conjugates for a functional property of the conjugate comprising simultaneously assaying a plurality of different peptide-cargo conjugates for said functional property.
  • screening of the peptide-cargo conjugates comprises use of the same cargo molecule together with the use of different peptides. In some embodiments of the method, screening of the peptide-cargo conjugates comprises use of different peptides together with different cargo molecules.
  • the method of screening may further comprise providing a library of different peptide- cargo conjugates on which to perform the assay.
  • the method of screening may further comprise generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
  • the method of screening may further comprise generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
  • a library of peptide-cargo conjugates comprising a plurality of containers each containing a quantity of an isolated or substantially purified peptide-cargo conjugate.
  • Each peptide-cargo conjugate is preferably different with respect to either the peptide or cargo component.
  • a peptide-cargo construct conjugate comprising a peptide bonded to a cargo construct comprising a cargo molecule and a releasable tag is provided, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide.
  • the peptide-cargo construct conjugate is preferably provided in isolated form.
  • each peptide-cargo construct conjugate comprises a peptide bonded to a cargo construct comprising a cargo molecule and a tag capable of association with a capture element on the solid support, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide molecule.
  • the peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates may comprise a cleavable linker attached to the tag and to the cargo molecule, the cleavable linker linking the tag and cargo molecule, preferably whereupon cleavage at (or of) the linker releases the peptide-cargo conjugate molecule from the tag.
  • a peptide-cargo construct conjugate comprising a peptide having a releasable tag, the peptide bonded to a cargo construct comprising a cargo molecule, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide.
  • each peptide-cargo construct conjugate comprises a peptide having a tag capable of association with a capture element on the solid support, the peptide bonded to a cargo construct comprising a cargo molecule, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide molecule.
  • the peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates the peptide may comprise a cleavable linker attached to the tag and to the peptide, the cleavable linker linking the tag and peptide, preferably whereupon cleavage at (or of) the linker releases the peptide- cargo conjugate molecule from the tag.
  • the peptide may have a maximum length of 60 amino acids and a minimum length of 2 amino acids. In some embodiments the peptide has a length selected from the group consisting of: 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 29 amino acids, 5 to 28 amino acids, 5 to 27 amino acids, 5 to 26 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 22 amino acids, 5 to 21 amino acids, or 5 to 20 amino acids.
  • the conjugates comprise a peptide covalently bonded to the cargo molecule.
  • the first and second functional groups form a pair having selective reactivity towards each other.
  • the first and second functional groups are selected from one of: an azide and an alkyne; a thiol and an alkene; a thiol and an alkyne; a diene and a dienophile; an isonitrile and a tetrazine; an epoxy and an aziridine; an amine and an isocyanate, an active methylene and an activated olefin; a thioester and a thiol (e.g.
  • one of the functional groups may need to be activated by prior reaction, for example where the first functional group is an amine and the second functional group is a carboxylic acid that is activated by reaction with a coupling agent such as HBTU in the presence of HOAt.
  • the first functional group is attached to the terminal amine of the peptide.
  • the cargo molecule is an oligonucleotide, an oligonucleotide analogue, a peptide or a peptide analogue.
  • the cargo molecule is a peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), or siRNA.
  • a method of peptide conjugate library synthesis is needed that is quick and efficient and is suitable for subsequent use of rapid screening assays. 4 Following cell assay of an initial library of conjugates using peptides of widely ranging sequence, fine-tuning of a peptide candidate can then be accomplished either by further narrower library synthesis and cell re-assay, or by more conventional synthesis if further screening is to be carried out by an in vivo assay.
  • the most time-limiting step in synthesis of peptide-cargo conjugates is generally not the conjugation itself but the individual purifications required for peptide, cargo and conjugate. Recently, parallel multi-peptide synthesis machines have become available as well as new methods for rapid affinity-based purification of bio-molecules (e.g.
  • biotidine tags or biotin-streptavidin 4b that provide an opportunity both for rapid synthesis and for reaction workup and purification.
  • bio-orthogonal "click"-type ligation reactions provide a chemical basis for efficient conjugation of bio-molecules in aqueous solution. 5
  • SELPEPCON PEPtide CONjugates
  • SELPEPCON allows for convenient and rapid parallel conjugation reactions and workup that avoid the need for HPLC purification ( Figure 1 ).
  • the method utilizes a functionalized cargo linked through a cleavable linker to an affinity tag.
  • the functionalized cargo is conjugated to the peptide after which it is purified by immobilization and isolated by release of the tag.
  • Affinity purification is very rapid and is readily automated, making the overall strategy very suitable for high-throughput synthesis of conjugates for screening.
  • peptide has its normally meaning in the art.
  • the term “peptide” includes a molecule comprising a contiguous sequence of amino acid residues where adjacent amino acids are joined by a peptide bond. As such, where the peptide is linear it normally has an N-terminus and a C-terminus, which are available for modification.
  • the peptide may contain amino acids of any type and in any sequence.
  • Amino acids may be natural or non-natural amino acids, D- or L- amino acids and may be modified amino acids.
  • non-natural amino acids include: 6-aminohexanoic acid (also called aminocaproic acid or ⁇ -aminocaproic acid or Ahx), 4-aminobutyric acid, aminocaprylic acid, ⁇ -alanine, p-aminobenzoic acid and isonipecotic acid.
  • Peptides according to the present invention may have a maximum length of 60 amino acid residues, or one of 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 residues.
  • Peptides according to the present invention may have a minimum length of 2 amino acid residues, or one of 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues.
  • peptides may have a length in the range of one of 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 29 amino acids, 5 to 28 amino acids, 5 to 27 amino acids, 5 to 26 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 22 amino acids, 5 to 21 amino acids, 5 to 20 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 29 amino acids, 6 to 28 amino acids, 6 to 27 amino acids, 6 to 26 amino acids, 6 to 25 amino acids, 6 to 24 amino acids, 6 to 23 amino acids, 6 to 22 amino acids, 6 to 21 amino acids, or 6 to 20 amino acids,
  • consideration of the length of the peptide does not include the length of the cargo molecule.
  • the peptide may be any kind of peptide. Preferably it is a linear peptide. In some embodiments it is a cell-penetrating peptide (CPP), or is a candidate cell-penetrating peptide.
  • the cargo molecule may be any small molecule, e.g. small molecule drug, peptide, cyclic peptide, protein, pharmaceutical or therapeutic (e.g. molecular weight less than one of 20,000 Da, 15,000 Da, 10,000 Da, 5,000 Da, 3000 Da or 1000 Da).
  • the cargo molecule may be a nucleic acid, antisense oligonucleotide, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA)), or siRNA.
  • PNA peptide nucleic acid
  • PMO phosphorodiamidate morpholino oligonucleotide
  • LNA locked nucleic acid
  • the cargo molecule is an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide, an oligonucleotide
  • oligonucleotide analogue a peptide or a peptide analogue.
  • Preferred oligonucleotide or peptide cargoes (or their analogues) are linear molecules, such as peptides,
  • Oligonucleotides or their analogues. Oligonucleotides may be single stranded or double stranded. Suitable oligonucleotide cargo molecules include conventional DNA and RNA
  • oligonucleotides which may include modified nucleotides, such as 2'modification of ribose, e.g. 2 -O-methyl (2'-OMe), 2'-amino (2'-NH) or 2'-Fluoro (2'-F) and/or 8-position purine modifications and/or 5-position pyrimidine modifications), LNA, siRNA.
  • modified nucleotides such as 2'modification of ribose, e.g. 2 -O-methyl (2'-OMe), 2'-amino (2'-NH) or 2'-Fluoro (2'-F) and/or 8-position purine modifications and/or 5-position pyrimidine modifications
  • LNA Long ribotide
  • siRNA siRNA
  • Suitable oligonucleotide analogues include PNA and PMO.
  • the cargo molecule is an oligonucleotide or oligonucleotide analogue it may have a maximum length of 5000 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs).
  • the maximum length may be one of 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs).
  • the minimum length may be one of 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs).
  • a "peptide-cargo conjugate” refers to a molecule in which a peptide, as described herein, is bonded (preferably covalently) to a cargo molecule, as described herein.
  • the covalent bond is preferably formed between the N- or C- terminus of the peptide and the cargo molecule.
  • the starting materials for reaction include a peptide attached to a first functional group and cargo molecule attached to a second functional group.
  • the attachment is preferably a covalent or ionic bond, most preferably a covalent bond.
  • the first and second functional groups form a pair having reactivity, preferably selective reactivity, towards each other such that upon contact a chemical reaction occurs leading to the formation of a covalent or ionic bond linking the peptide and cargo molecule.
  • the reaction may be subject to catalysis.
  • a range of functional groups are available, including pairs of functional groups that are bio-orthogonal, having selective reactivity towards each other.
  • the first and second functional groups may form a pair of functional groups having reactivity using Click chemistry.
  • Click chemistry is preferred because typical Click chemistry reactions are simple, require simple or no purification and give high yields.
  • Click chemistry reactions include cycloadditions and additions to double or triple bonds. More specific examples include [3+2] cycloadditions, such as the Cu(l)-catalyzed stepwise Huisgen 1 ,3-dipolar cycloaddition, thiol-alkene and thiol alkyne click reactions, Diels-Alder reactions and inverse electron demand Diels-Alder reactions,
  • [4+1] cycloadditions nucleophilic substitution especially to small strained rings, non-aldol type carbonyl reactions, such as the formation of ureas, and Michael Additions.
  • Other examples include native chemical ligation of a thiolate group of an N-terminal cysteine with a C-terminal thioester, hydrazide formation by reaction of an aldehyde with a hydrazine, or oxime formation by reaction of an aldehyde with an aminooxy derivative, e.g. a hydroxylamine.
  • suitable first and second functional group pairs include: an azide and an alkyne; a thiol and an alkene; a thiol and an alkyne; a diene and a dienophile; an isonitrile and a tetrazine; an epoxy and an aziridine; an amine and an isocyanate, an active methylene and an activated olefin, a thioester and a thiol; an aldehyde and a hydrazine; an aldehyde and an aminoxy derivative.
  • Examples of each functional group suitable for the Click chemistry reactions are known in the art.
  • a particularly preferred functional group pair is an azide and an alkyne.
  • Azides and alkynes may be used in the Huisgen 1 ,3-dipolar cycloaddition. The reaction forms a 1 ,2,3-triazole link.
  • the alkyne may be a terminal or internal alkyne. The reaction of the azide with the alkyne may be catalysed.
  • the reaction may be a Copper(l)- catalyzed azide-alkyne cycloaddition (CuAAC), a Ruthenium-catalysed 1 ,3-dipolar azide- alkyne cycloaddition (RuAAC) or a Silver(l)-catalyzed azide-alkyne cycloaddition
  • CuAAC Copper(l)- catalyzed azide-alkyne cycloaddition
  • RuAAC Ruthenium-catalysed 1 ,3-dipolar azide- alkyne cycloaddition
  • silver(l)-catalyzed azide-alkyne cycloaddition a Silver(l)-catalyzed azide-alkyne cycloaddition
  • reaction may be uncatalysed, e.g. where the alkyne is cylooctyne.
  • the reaction conditions for such reactions are known in the art.
  • the choice of which functional group is the first functional group on the peptide or the second functional group on the cargo molecule is not particularly limited. The choice may be determined, for example, by ease of preparation of the peptide and cargo molecule and/or the compatibility of the functional group with other functional groups in the peptide or cargo molecule.
  • an isocyanate may not be suitable as the first functional group because of potential inter- or intra-molecular reaction with peptide amine groups.
  • the first functional group is an alkyne and the second functional group is an azide.
  • the method of the present invention preferably includes a Copper(l)-catalyzed azide-alkyne cycloaddition (CuAAC) to link the peptide to the cargo molecule.
  • the peptide-cargo conjugate contains a 1 ,2,3-triazole link.
  • the alkyne functionality is preferably located on the peptide and the azide functionality on the cargo.
  • the functional group may be attached to the peptide at any suitable position, but is preferably attached to the peptide at the N-terminus (preferably via bonding to the nitrogen of the terminal amine) or C-terminus (preferably via bonding to the carbon of the terminal carboxyl), and most preferably at the N-terminus via bonding to the nitrogen of the terminal amine.
  • the functional groups may be incorporated in the peptide and/or cargo molecules during synthesis, e.g. as part of the final coupling step in peptide or oligonucleotide synthesis.
  • Attachment of the functional group to the cargo molecule may be at any suitable position. Where the cargo molecule is a linear molecule, attachment is preferably at one end of the molecule.
  • attachment may be at the N- or C- terminus, as described above.
  • N-terminal attachment is preferred. This may provide for a conjugate in which the N-terminus of the peptide is conjugated to the N- terminus of the cargo peptide.
  • a conjugate is formed in which the N-terminus of the peptide is conjugated to the C-terminus of the cargo peptide.
  • a conjugate is formed in which the C-terminus of the peptide is conjugated to the C-terminus of the cargo peptide.
  • the functional group may be attached at either the 3' or 5' end.
  • attachment of the functional group may involve modification of the peptide or cargo to facilitate attachment of the functional group.
  • the cargo molecule (or optionally the peptide) comprises a tag which is preferably attached to the cargo molecule or part of the cargo construct by a covalent or ionic bond.
  • the tag is preferably releasable from the cargo molecule.
  • the cargo molecule (or optionally the peptide) is also attached to a cleavable linker.
  • the cleavable linker is preferably attached to the tag and to the cargo molecule (or optionally the peptide), and may be positioned between the tag and cargo molecule (or optionally the peptide). Attachment of the cleavable linker to the tag and/or cargo molecule (or optionally the peptide) is preferably by covalent and/or ionic bond.
  • the cargo construct comprises a linear molecule having a tag at one end attached to a cleavable linker which is attached to the cargo molecule (which may also be a linear molecule).
  • the cleavable linker may be positioned between the tag and cargo molecule.
  • the cargo construct may optionally have a linear arrangement of tag-cleavable linker-cargo molecule, in such an arrangement the cleavable linker being called an in-line cleavable linker. Cleavage of the cleavable linker leads to detachment/release of the tag (and optionally all or part of the cleavable linker) from the cargo construct.
  • Cleavable linkers are available that permit selective and controlled cleavage by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents [Leriche et al (Cleavable linkers in chemical biology. Bioorg Med Chem. 2012 Jan 15;20(2):571-82)].
  • peptide-cargo construct conjugates are partitioned by capturing or immobilizing on a solid support using a tag incorporated into the cargo construct.
  • a tag incorporated into the cargo construct.
  • beads having a capture element such as avidin, streptavidin, neutravidin, or extravidin, can be used to capture the peptide-cargo construct conjugates. The beads maybe washed to remove any free peptide.
  • a tag refers to a component that provides a means for attaching or immobilizing a cargo construct (and any peptide conjugated to it) to a solid support.
  • a tag is a component that is capable of associating with a capture element.
  • a tag can be attached to or included in the cargo construct by any suitable method.
  • the tag allows the cargo construct to associate, either directly or indirectly, with a capture element that is attached to a solid support.
  • the capture element is typically chosen (or designed) to be highly specific in its interaction with the tag and to retain that association during subsequent processing steps or procedures.
  • a tag can enable the localization of a cargo construct (or peptide-cargo construct conjugate) to a spatially defined address on a solid support.
  • a tag can be a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, biotin, polyhistidine, or any fragment or derivative of these structures, any combination of the foregoing, or any other structure with which a capture element can be designed or configured to bind or otherwise associate with specificity.
  • a tag is configured such that it does not interact intra-molecularly with either itself or the cargo construct to which it is attached or of which it is a part.
  • the tag is biotin group and the capture element is a biotin binding protein such as avidin, streptavidin, neutravidin, or Extravidin.
  • a biotin binding protein such as avidin, streptavidin, neutravidin, or Extravidin. This combination may be conveniently used in various embodiments, as biotin is easily incorporated into cargo constructs during synthesis and streptavidin beads are readily available.
  • the tag is polyhistidine and the capture element is nitrilotriacetic acid (NTA) chelated with a metal ion such as nickel, cobalt, iron, or any other metal ion able to form a coordination compound with poly-histidine when chelated with NTA.
  • NTA nitrilotriacetic acid
  • a tag can associate directly with a probe on the surface of the solid support and covalently bind to the probe, thereby covalently linking the cargo construct to the surface of the solid support.
  • the tag and the probe can include suitable reactive groups that, upon association of the tag with the probe, are sufficiently proximate to each other to undergo a chemical reaction that produces a covalent bond.
  • the reaction may occur spontaneously or may require activation, such as, for example, photo-activation or chemical activation, for example in one embodiment where the first functional group is an amine and the second functional group is a carboxylic acid that is activated by reaction with a coupling agent such as HBTU in the presence of HOAt so that an amide bond is formed.
  • the tag includes a diene moiety and the probe includes a dienophile, and covalent bond formation results from a spontaneous Diels-Alder conjugation reaction of the diene and dienophile. Any appropriate
  • complementary chemistry can be used, such as, for example, N-Mannich reaction, disulfide formation, native ligation, hydrazide formation, oxime formation, Curtius reaction, Aldol condensation, Schiff base formation, and Michael addition.
  • disulfide bond is the cleavable linker the reaction between tag and probe preferably does not rely on disulfide bond formation.
  • the tag component is bi-functional in that it includes functionality for specific interaction with a capture element on a solid support and functionality for dissociating the cargo molecule to which it is attached from the tag.
  • the means for dissociating the tag includes chemical means, photochemical means or other means depending upon the particular tag that is employed.
  • a capture element refers to a molecule that is configured to associate, either directly or indirectly, with a tag.
  • a capture element is a molecule or type of multi-molecular structure that is capable of immobilizing the cargo molecule to which the tag is attached to a solid support by associating, either directly or indirectly, with the tag.
  • a capture element can be a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, bitoin, polyhistidine, or any fragment or derivative of these structures, any combination of the foregoing, or any other structure with which a tag can be designed or configured to bind or otherwise associate with specificity.
  • a capture element can be attached to a solid support either covalently or non-covalently by any suitable method.
  • a tag in one embodiment may be used as a capture element in another embodiment, and a capture element in one embodiment may be used as a tag in another embodiment.
  • a cargo construct with a biotin tag may be captured with streptavidin attached to a solid support in one embodiment, while a cargo construct with a streptavidin tag may be captured with biotin attached to a solid support in another embodiment.
  • a releasable or cleavable element, moiety, or linker refers to a molecular structure that can be broken to produce two separate components.
  • a releasable (or cleavable) element may comprise a single molecule in which a chemical bond can be broken, or it may comprise two or more molecules in which a non-covalent interaction can be broken or disrupted.
  • oligonucleotide cargo molecule it is necessary to spatially separate certain chemical groups or moieties from others in order to prevent interference with the individual functionalities or to provide for the attachment of a functional group.
  • Spacing linkers may be used for this purpose.
  • Spacing linkers may be introduced into an oligonucleotide, PNA, PMO or peptide cargo molecules during synthesis and so can be comprised of number of phosphoramidite spacers, including but not limited to aliphatic carbon chains of length 3, 6, 9, 12 and 18 carbon atoms, polyethylene glycol chains of length 1 , 3, and 9 ethylene glycol units, or a tetrahydrofuran moiety (termed dSpacer (Glen Research) or any combination of the foregoing or any other structure or chemical component that can be designed or configured to add length along a phosphodiester backbone.
  • dSpacer Glen Research
  • the spacing linker includes polynucleotides, such as poly dT, dA, dG, or dC or poly U, A, G, or C or any combination of the foregoing.
  • spacers include one or more abasic ribose or deoxyribose moieties. Spacing linkers may also be included at one or both ends of a cargo molecule, including oligonucleotide or peptide cargo molecules. Suitable spacing molecules include those described above. For peptide cargoes, spacing molecules may also include natural or non-natural amino acids, D- or L- amino acids or ethylene glycol or polyethylene glycol spacers. Examples of suitable non-natural amino acids include: 6-aminohexanoic acid, 4-aminobutyric acid, aminocaprylic acid, ⁇ -alanine, p-aminobenzoic acid and isonipecotic acid.
  • An in-line cleavable linker refers to a group of atoms that contains a releasable or cleavable element.
  • an in-line cleavable linker is used to join a cargo molecule to a tag, thereby forming a releasable tag.
  • An in-line cleavable linker may be chemically cleavable in that it includes a bond that can be cleaved by treating it with an appropriate chemical or enzymatic reagent.
  • a disulphide bond that can be cleaved by treating it with a reducing agent to disrupt the bond.
  • An in-line cleavable linker may be photo-cleavable (in that it includes a bond that can be cleaved by irradiating the releasable element at the appropriate wavelength of light).
  • a solid support refers to any substrate having a surface to which molecules may be attached, directly or indirectly, through either covalent or non-covalent bonds.
  • the solid support may include any substrate material that is capable of providing physical support for the capture elements or probes that are attached to the surface. The material is generally capable of enduring conditions related to the attachment of the capture elements or probes to the surface and any subsequent treatment, handling, or processing encountered during use.
  • the materials may be naturally occurring, synthetic, or a modification of a naturally occurring material.
  • Suitable solid support materials may include silicon, graphite, mirrored surfaces, laminates, ceramics, plastics (including polymers such as, e.g., polyvinyl chloride), cyclo-olefin copolymers, agarose gels, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene ), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon, polyvinyl butyrate)), germanium, gallium arsenide, gold, silver.
  • plastics including polymers such as, e.g., polyvinyl chloride), cyclo-olefin copolymers, agarose gels, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene ), polystyrene, polyme
  • Additional rigid materials may be considered, such as glass, which includes silica and further includes, for example, glass that is available as Bioglass.
  • Other materials that may be employed include porous materials, such as, for example, controlled pore glass beads, crosslinked beaded sepharose or agarose resins, or copolymers of crosslined
  • the material used for a solid support may take any of a variety of configurations ranging from simple to complex.
  • the solid support can have any one of a number of shapes, including a strip, plate, disk, rod, particle, bead, tube, well, or column.
  • the solid support may be porous or non-porous, magnetic, paramagnetic, or non-magnetic, polydisperse or monodisperse, hydrophilic or hydrophobic.
  • the solid support may also be in the form of a gel or slurry of closely packed (as in a column matrix) or loosely-packed particles.
  • the solid support with attached capture element may be used to capture tagged peptide- cargo construct conjugates.
  • the solid support could be a streptavidin-coated bead or resin such as Dynabeads M-280
  • partition refers to separation or removal of one or more molecular species, complexes or conjugates. Partitioning of free peptide from peptide- cargo construct conjugates is effective following formation of peptide-cargo construct conjugates.
  • the partitioning step may separate peptide-cargo construct conjugates bound to a capture element, thereby forming a complex of cargo construct and capture element, from peptide not bound to capture element.
  • Partitioning may involve immobilization of peptide-cargo conjugates on a solid support thereby separating and purifying the conjugates. Partitioning may also involve separation of peptide-cargo conjugates from unwanted contaminants.
  • the methods of forming peptide-cargo conjugates or generating peptide libraries according to this disclosure may comprise one or more optional wash steps, e.g. after formation of peptide-cargo constructs, or following partition of reagents/products.
  • the release of peptide-cargo conjugate from the tag refers to the separation of the conjugated cargo molecule and peptide from the tag and any capture element with which it is associated. Release may involve cleavage of the tag and/or cleavabie linker to separate the peptide-cargo molecule construct from the tag and/or cleavabie linker. Release may therefore involve a step of cleavage of the peptide-cargo conjugate from the tag and/or linker. The release may provide free peptide-cargo conjugates, which may be collected, isolated and/or purified to provide a composition comprising isolated and/or substantially purified peptide-cargo conjugates.
  • a plurality refers to at least two, but preferably refers to one of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 1 1 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more.
  • synthesis reactions are performed in parallel. This refers to performing a plurality of synthesis reactions simultaneously thereby allowing for the generation of a plurality of different peptide-cargo conjugates, or peptide-cargo construct conjugates in about the time required to complete a single synthesis reaction.
  • Parallel reactions may be performed side-by-side, e.g. in a plurality of adjacent reaction chambers.
  • a multi-well plate e.g. 12, 48 or 96 well plate may be used in which individual reactions are performed in separate wells of the plate.
  • Synthesis reactions or screening may be automated, e.g. the method, experiment or reaction may be performed or conducted without manual human intervention being performed by a machine or robot under the control of a suitably programmed computer.
  • reactions or assays are performed simultaneously. This refers to performing the reactions or assays at the same time, rather than consecutively, such that a plurality of reactions or assays may be completed in about the time required to complete a single reaction or assay. This permits the high-throughput synthesis of conjugates and high-throughput screening of conjugates required during the process of drug discovery or validation.
  • assays of peptide-cargo conjugates are performed in respect of a functional property.
  • the functional property may be any such property of interest.
  • the assay may test the ability of the peptide to act as a cell penetrating peptide (CPP) [a CPP screening assay] in combination with its cargo.
  • CPP cell penetrating peptide
  • Such assays are well known (e.g. the HeLa pLuc705 cell assay described herein) and generally test for the ability of the peptide to facilitate uptake of the cargo molecule into a cell. The uptake may be assessed against a control comprising the cargo molecule not conjugated to the peptide.
  • a library refers to a collection of different peptide-cargo conjugates, each stored individually, e.g. in an individual container, but also as part of a collection in which the plurality of peptide-cargo conjugates/containers are stored in one geographical location, e.g. one or more cold stores (fridges, freezers) in the same building.
  • Each container may contain a sufficient quantity of isolated or purified peptide- cargo conjugate to permit a plurality of samples to be taken, e.g. to allow use in multiple function assays or screening methods.
  • each container may contain a quantity of peptide-cargo conjugate in the range of 10-1000ng or 1-1000Mg or 1-1 Omg.
  • the library may contain a plurality of series' of conjugates. Each series comprises a plurality of peptide-cargo conjugates, as described above. Within a given series the cargo molecule is the same for each conjugate but the peptide is different between each conjugate. Between series the cargo molecule is different and the conjugates within a series differ in respect of the peptide component and preferably have a range of peptide components that corresponds to the other series in the set.
  • a library may have one or a plurality of different sets of series of conjugates, e.g. the sets differing in terms of the composition of the peptide components.
  • compositions comprising isolated and/or purified or substantially purified peptide-cargo construct conjugates or peptide- cargo conjugates are provided.
  • Such compositions are enriched for the respective conjugate, and may be free or substantially free of unreacted peptide (e.g. less than one of 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of the peptide component of the composition is free or unreacted compared with the conjugated peptide component) and/or of unreacted cargo.
  • unreacted peptide e.g. less than one of 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of the peptide component of the composition is free
  • Methods according to the present invention are preferably performed in vitro.
  • the term "in vitro" is intended to encompass experiments with reagents, reactants, materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term “in vivo” is intended to encompass experiments and procedures with intact multicellular organisms.
  • the method may comprise a high throughput screening assay.
  • Test compounds used may be obtained from a synthetic combinatorial peptide library, or may be synthetic peptides or peptide mimetic molecules. Other test compounds may comprise defined chemical entities, oligonucleotides or nucleic acid ligands.
  • a screening assay may be performed in vivo where only a small quantity (e.g. ⁇ gs) of conjugate is required to be delivered (e.g. by intramuscular delivery) into an animal such as a mouse.
  • the peptide is a cell penetrating peptide (CPP;
  • Cell penetrating peptides are peptides that are useful in facilitating the uptake of a conjugated cargo molecule, e.g. where the cargo molecule is useful as a therapeutic but requires uptake to a cell in order to exert its therapeutic effect.
  • oligonucleotide analogues useful as steric blocking agents such as those with uncharged backbones, such as peptide nucleic acids (PNA) [Egholm M., Buchardt O., Nielsen P. E., Berg R. H.: J. Amer. Chem. Soc. 1992, 114, 1895] and phosphorodiamidate morpholino oligonucleotides (PMO) [Summerton J., Weller D.: Antisense Nucl. Acid Drug Dev. 1997, 7, 187]. Both PNA and PMO ONs have been used in vivo for RNA targeting applications towards the
  • a range of CPPs are known. Although some known CPPs share common sequence structure characteristics, a single set of such characteristics does not define a CPP per se.
  • a candidate CPP may have any amino acid sequence. Indeed, the present invention is useful in investigating sequence structure of new candidate CPPs as well as in investigating sequence variation of known CPPs.
  • Known CPPs are described in WO201 1/064552 WO2009144481 , WO2009/147368 and WO2013/030569, each of which are incorporated by reference in their entirety.
  • known CPPs include Penetratin, Tat, Transportan, and (R-Ahx-R) 4
  • Rhx aminohexanoic acid
  • R6Pen R6-Penetratin
  • a class of CPP can be defined as a peptide having a primary sequence structure comprised of at least three domains, having the arrangement:
  • the number of R (Arginine) residues in Domains 1 and 3 combined is at least 5
  • the number of X residues in Domains 1 and 3 combined is at least 1
  • the number of B residues in Domains 1 and 3 combined is at least 2
  • X one of 6-aminohexanoic acid, 4-aminobutyric acid, aminocaprylic acid, p-aminobenzoic acid or isonipecotic acid
  • B betaAlanine
  • Domain 2 comprises a sequence that contains at least 3 of the amino acids Z1Z2FLI, where Zi is Y or I and Z2 is Q or R.
  • a class of CPP can be defined as a peptide having a primary sequence structure comprised of at least three domains, having the arrangement::
  • Domain 1 comprises a sequence chosen from RXRRBRRXR, RBRRXRRBR, RXRRXR
  • Domain 2 comprises a sequence chosen from: ILFQ, ILIQ
  • a class of CPP can be defined as a peptide having a primary sequence structure comprised of at least three domains, having the arrangement:
  • Domain 1 comprises a sequence chosen from RXRZ3 where Z3 is selected from one of: RBRRXR, RBRRX, RBRX, RBRXR, BRX, BX, RBR, RB, or RBRR
  • Domain 2 comprises a sequence that contains at least 3 of the amino acids Z-
  • CPPs include MSP (ASSLNIA), AAV6
  • TVAVNLQSSSTDPATGDVHVM the AAV8 IVADNLQQQNTAPQIGTVNSQ, TAT (YGRKKRRQRRRP) or (RXR)4 (RXRRXRRXRRXR) [where R is L-arginine and X is 6- aminohexanoic acid], and suitable peptides may comprise the sequences.
  • An aminoalkyi spacer is typically a molecule that can separate amino acids in the peptide chain.
  • the aminoalkyi spacer may have from 1 to 6, such as 2, 3, 4 or 5, carbon atoms.
  • the aminoalkyi spacer typically comprises an amino group and a carboxyl group such that it can bind to the adjacent amino acids in the peptide chain through peptide bonds.
  • Preferred aminoalkyi spacers include, but are not limited to, 6-aminohexanoyl (X), betaalanyl (B), 4-aminobutyryl, p-amino benzoyl, or isonipecotyl.
  • a candidate CPP may comprise two or more RZR groups (for example RXR and/or RBR groups). The number of these groups being determined by the value of n, where n is from 2 to 6, such as 3, 4 or 5.
  • the Z in RZR may independently be X or B.
  • the peptide may comprise the sequence RXRRXR, RBRRBR, RXRRBR or RBRRXR.
  • the two or more RXR and/or RBR groups may be separated by Z (if I is 1 ) and/or ILFQY (if m is 1 ).
  • n if I is 1 , m is preferably 0.
  • I is preferably 0.
  • the separating group may independently be Z or ILFQY.
  • the peptide may comprise the sequence RXRZRXRILFQYRXR (i.e. where the first two RZRs are separated by Z and the second two RZRs are separated by ILFQY).
  • the invention concerns the provision and/or formation and/or screening of a peptide-cargo construct comprising a cell delivery peptide (the peptide), or candidate cell delivery peptide, covalently or non-covalently attached to a biologically active compound (the cargo).
  • Peptide-cargo conjugates according to the present invention may be provided for use in a method of medical treatment.
  • the medical treatment may preferably require delivery of the cargo molecule into a cell and optionally the nucleus of the cell.
  • Peptides and/or peptide-cargo conjugates are accordingly provided for use in treatment of disease.
  • the use of a peptide and/or a peptide-cargo conjugate in the manufacture of a medicament for the treatment of disease is also provided.
  • a method of treatment of a patient or subject in need of treatment for a disease condition is also provided comprising the step of administering a therapeutically effective amount of a peptide and/or a peptide- cargo conjugate to the patient or subject.
  • the cargo component of a peptide- cargo conjugate comprises an active agent (e.g. pharmaceutical agent) capable of treating, preventing or ameliorating the disease.
  • Diseases to be treated may include any disease where improved penetration of the cell and/or nuclear membrane by a pharmaceutical or therapeutic molecule may lead to an improved therapeutic effect.
  • Diseases to be treated may include disease conditions caused by (in whole or in part) splicing deficiencies, e.g. Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy, and other muscle diseases such as limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculpharyngeal muscular dystrophy (OMD), distal muscular dystrophy and Emery-Dreifuss muscular dystrophy (EDMD), as well as Menkes
  • DMD Duchenne Muscular Dystrophy
  • LGMD limb-girdle muscular dystrophy
  • OMD oculpharyngeal muscular dystrophy
  • EDMD Emery-Dreifuss muscular dystrophy
  • ⁇ -thalassemia 39 splice correction of tau protein to relieve frontotemporal dementia, parkinsonism and spinal muscular atrophy 39 , Hutchinson-Gilford Progeria Syndrome 40 , Ataxia-telangiectasia mutated (ATM) 41 , spinal muscular atrophy, myotonic dystrophy 1 , or cancer.
  • ATM Ataxia-telangiectasia mutated
  • dystrophin Duchenne muscular dystrophy and Becker muscular dystrophy
  • DMPK DM1 type MD
  • ZNF9 DM2 type MD
  • PABPN1 PABPN1
  • emerin lamin A or lamin C
  • EDMD myotilin
  • LGMD-1A myotilin
  • LGMD-1 B myotilin
  • LGMD-1 C caveolin-3
  • LGMD-2A dysferlin
  • LGMD-2C alpha-sarcoglycan
  • LGMD-2D beta-sarcoglycan
  • LGMD-2F and CMD1 L telethonin
  • LGMD-2G TRIM32
  • fukutin-related protein LGMD-2I
  • titin LGMD-2J
  • the cargo may comprise an oligonucleotide, PNA, PMO or other oligonucleotide types, including LNA, capable of preventing or correcting the splicing defect and/or increasing the production of (e.g. number of) correctly spliced mRNA molecules.
  • the present invention is, of course, not limited to cargo molecules capable of correcting a splicing defect.
  • Cargo molecules may include other oligonucleotide, PNA, PMO or LNA molecules such as oligonucleotide molecules capable of targeting mRNA or microRNA, e.g.
  • the patient or subject to be treated may be any animal or human.
  • the patient or subject may be a non-human mammal, but is more preferably a human patient.
  • the patient or subject may be male or female.
  • Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural,
  • the medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for
  • compositions which may be based on a peptide-cargo conjugate formed, screened, assayed or tested as described herein.
  • methods of production may further comprise one or more steps selected from:
  • a further aspect of the present invention relates to a method of formulating or producing a pharmaceutical composition, the method comprising identifying a peptide- cargo conjugate in accordance with one or more of the methods described herein, and further comprising one or more of the steps of:
  • compositions formulated by such methods may comprise a prodrug of the selected substance wherein the prodrug is convertible in the human or animal body to the desired active agent.
  • the active agent may be present in the pharmaceutical composition so produced and may be present in the form of a physiologically acceptable salt.
  • the designing of mimetics to a known pharmaceutically or biologically active compound is a known approach to the development of pharmaceuticals and therapeutics based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. some peptides may be unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • the physical properties e.g. stereochemistry, bonding, size and/or charge
  • data from a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • a method comprising the step of modifying the peptide structure and conjugating to a peptide or oligonucleotide cargo, such as PNA or PMO, optionally followed by testing the modified peptide-cargo conjugate in a splice correction assay or an exon skipping assay and/or in a cell viability assay.
  • This process of modification of the peptide or peptide mimetic conjugate may be repeated a number of times, as desired, until a peptide-cargo conjugate having the desired splice correction or exon skipping activity and/or cell viability is identified.
  • the modification steps employed may comprise truncating the peptide or peptide mimetic length (this may involve synthesising a peptide or peptide mimetic of shorter length), substitution of one or more amino acid residues or chemical groups, and/or chemically modifying the peptide or peptide mimetic to increase cell viability of its corresponding cargo conjugate, resistance to degradation, transport across cell membranes and/or resistance to clearance from the body and/or to provide activity in exon skipping and dystrophin production in an mdx mouse model of DMD, including in heart muscle.
  • the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
  • Figure 1 Diagrammatic overview of conjugation and conjugate work-up strategy of SELPEPCON.
  • Figure 2 Chart showing fold increase in luminescence caused by PNA705 conjugates of LB 1 -peptides compared to a buffer blank induced by the conversion of Beetle luciferin to oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 ⁇ conjugate concentration.
  • Figure 3 Chart showing fold increase in luminescence caused by PNA705 conjugates of LB2-peptides compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 ⁇ conjugate concentration.
  • Figure 4 Chart showing fold increase in luminescence caused by PNA705 conjugates of LB2-peptides obtained by SELPEPCON (Crude) and RP-HPLC purified (Purified) compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 ⁇ conjugate concentration.
  • Chart showing fold increase in luminescence as a function of a) the net charge and b) the number of arginines of the CPPs in the LB2-PNA705 conjugates compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 ⁇ conjugate concentration.
  • the dots represent the data points and the line represents the average.
  • FIG. 6 RT-PCR analysis to measure aberrant and redirected RNA levels in HeLa pLuc705 cells after exposure to CPP-PNA705 conjugates at different concentrations. The activity is presented as a percentage of corrected RT-PCR product obtained from splicing redirection.
  • Scheme 2 Synthesis of alkyne functionalized peptides for conjugation to N 3 -PNA705-S-S-biotin Figure 9.
  • Scheme 3 Outline of conjugation and purification strategy to obtain CPP- PNA705 conjugate libraries Figure 10.
  • Table 1 Library of Pip5h analogues containing an A/-terminal alkyne linker, peptide and conjugate synthesis yield.
  • Table 2 MALDI-TOF mass-spectrometry analysis of LB1 -peptides and their PNA705 conjugates synthesized using SELPEPCON
  • Table 3 Sequences and MALDI-TOF mass-spectrometry analysis of LB2 CPP-library containing a A erminal alkyne linker and conjugation to N3-PNA705-S-S- biotin.
  • Figure 13 Table 4: Sequences and MALDI-TOF mass-spectrometry analysis of LB2 CPP-library containing a /V-terminal alkyne linker and conjugation to N3-PNA705-S-S- biotin.
  • Table 5 Sequences and MALDI-TOF mass-spectrometry analysis of LB2 CPP-library containing an A/-terminal alkyne linker and conjugation to N 3 -PNA705-S-S- biotin.
  • Table 8 Yields of parallel peptide and PNA705-conjugate synthesis of LB2-CPP-Library.
  • FIG. 18 MALDI mass-spectra of parallel-synthesized LB1 - and LB2-peptides obtained by SELPEPCON.
  • A MALDI-TOF mass-spectra of LB1 - and LB2-peptides obtained using the SELPEPCON parallel synthesis procedure.
  • K and (L) show peptides that showed several peaks belonging to impurities.
  • A MALDI- TOF mass-spectrum of LB1_1 .
  • B MALDI-TOF mass-spectrum of LB1_12.
  • C MALDI- TOF mass-spectrum of LB2_3.
  • D MALDI-TOF mass-spectrum of LB2_5.
  • E MALDI- TOF mass-spectrum of LB2_7.
  • F MALDI-TOF mass-spectrum of LB2_24.
  • G MALDI- TOF mass-spectrum of LB2_27.
  • H MALDI-TOF mass-spectrum of LB2_54.
  • I MALDI- TOF mass-spectrum of LB2_68.
  • J MALDI-TOF mass-spectrum of LB2_72.
  • K DI-TOF mass-spectrum of LB2_40.
  • L MALDI-TOF mass-spectrum of LB2_5 .
  • FIG. 19 HPLC graphs of parallel-synthesized peptides obtained by SELPEPCON. These were recorded on a Phenomenex analytical C18 Jupiter column (250 x 4.6 mm, 5 micron) using the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 15% B 2-20 min 15%-30% B 20-25 min 30%-90% B.
  • A HPLC-graph of the LB1_5 obtained by SELPEPCON.
  • B HPLC-graph of the LB1_10 obtained by SELPEPCON.
  • FIG. 20 MALDI mass-spectra of LB1 - and LB2-PNA705 conjugates obtained by SELPEPCON.
  • A) to (M) examples are given of MALDI-TOF mass-spectra of conjugates obtained using the SELPEPCON procedure.
  • (A) to (L) show MALDI-TOF mass-spectra of PNA705 conjugates from libraries LB1 and LB2 that were tested in the splicing redirection assay.
  • Figure (M) shows a MALDI-TOF mass- spectrum of a conjugate for which the conversion of the conjugation reaction was low and was thus not tested.
  • Figure (K) shows an example of a conjugate containing a cysteine in the CPP-sequence for which the mixture of conjugate containing different amounts of StBu groups was found by MALDI-spectroscopy.
  • A MALDI-TOF mass-spectrum of the PNA705 conjugate of LB1_4.
  • B MALDI-TOF mass-spectrum of the PNA705 conjugate of LB1_8.
  • C MALDI-TOF mass-spectrum of the PNA705 conjugate of LB1_1 1.
  • D MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_27.
  • E MALDI-TOF mass- spectrum of the PNA705 conjugate of LB2_28.
  • F MALDI-TOF mass-spectrum of the
  • PNA705 conjugate of LB2_35 (G) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_38. (H) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_43. (I) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_44. (J) MALDI-TOF mass- spectrum of the PNA705 conjugate of LB2_54. (K) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_68. (L) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_71. (M) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_31.
  • FIG. 21 HPLC graphs of CPP-PNA705 conjugates obtained by SELPEPCON.
  • a and B are example HPLC graphs of LB2-PNA705 conjugates. These were recorded on a Phenomenex analytical C18 Jupiter column (250 x 4.6 mm, 5 micron) using the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 10% B 2-20 min 10%-30% B 20-30 min 30%-50% B 30-35 min 50%-90% B.
  • Unconjugated PNA impurities ( ⁇ 10%) can be observed which are likely the results of some impurities in the PNA that do not contain the azide functional group.
  • Figure 22 Chart showing fold increase in luminescence caused by PNA705 conjugates of LB1 -peptides obtained by SELPEPCON (Crude) and RP-HPLC purified (Purified) compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 1 and 2 ⁇ conjugate concentration.
  • Figure 23 Chart showing fold increase in luminescence caused by PNA705 conjugates of LB1 -peptides obtained by SELPEPCON (Crude) and RP-HPLC purified (Purified) compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 1 and 2 ⁇ conjugate concentration.
  • Figure 23 Chart showing fold increase in luminescence caused by PNA705 conjugates of LB1 -peptide
  • Figure 25 Table 10 MALDI-TOF mass-spectrometry analysis of C-terminal alkyne containing LB -peptides and their PNA705 conjugates synthesized using SELPEPCON.
  • Figure 27 Table 12. Conjugation of LB2 CPP-peptides containing an /V-terminal alkyne linker to azide modified PMI constructs.
  • Figure 28 Diagram illustrating synthesis of bifunctional PMO-cargo.
  • FIG. 29 Diagram illustrating the conjugation of biotinylated peptides containing a CXB C-terminal extension to PMO via SELPEPCON and the subsequent capping of conjugates using iodoacetamide.
  • SELPEPCON SELPEPCON methodology
  • two case studies involving a PNA cargo are shown.
  • a small conjugate library is synthesized to investigate the roles of individual or groups of amino acids in a pre-selected CPP attached to a cargo.
  • the second shows a larger CPP screen of a variety of quite different peptide sequences to find CPP candidates suitable for delivery of a novel cargo into a cell.
  • CCTCTTACCTCAGTTACA CCTCTTACCTCAGTTACA
  • a disulphide-linked biotin tag allows for solid-phase immobilization purification of the resultant conjugates after which the conjugates can be isolated by reduction of the disulphide, which releases it from the solid support.
  • the results demonstrate how SELPEPCON can be utilized to find active CPPs for a cargo such as PNA705 in a rapid synthesis, isolation and screening procedure.
  • MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro
  • the PNA705 sequence containing a C-terminal Cys and two flanking Lys residues was synthesised on a 50 ⁇ scale using a modified Liberty Peptide Synthesizer (CEM) according to a published procedure [10] using a Chem-Matrix solid support and Fmoc amino acid monomers (Novabiochem) or Fmoc (Bhoc) PNA monomers (Link Technologies). After the final deprotection, the support was removed from the Synthesizer and 5-azidopentanoic acid was manually double coupled using a standard PyBop/NMM coupling reaction at room temperature for 2 x 15 min.
  • CEM modified Liberty Peptide Synthesizer
  • the PNA was cleaved from the support and deprotected using TFA (1.5 mL) containing 10% triisopropylsilane/2.5% water and 1 % phenol. The mixture was agitated for 2 h after which it was filtered and concentrated. The crude PNA was isolated by cold diethyl ether precipitation, dissolved in water, filtered and purified by HPLC.
  • Peptide synthesis Peptide library synthesis was carried out on a 5 prnol scale using an Intavis Parallel Peptide Synthesizer, applying standard Fmoc chemistry and following manufacturer's recommendations.
  • the solid support was as supplied by Intavis (Tentagei, 0.2 mmol g ). Double coupling steps were used with a PyBop/NMM coupling mixture followed by acetic anhydride capping after each coupling step.
  • Terminal alkyne functionalization involved a standard coupling procedure using 4-pentynoic acid in the final step of the synthesis.
  • the peptides were cleaved from the support and deprotected by addition of TFA (1.5 mL) containing 5% triisopropylsilane/2.5% water and 1 % phenol with shaking for 4 h.
  • TFA 1.5 mL
  • the support suspension was then concentrated to a volume of ⁇ 500 ⁇ _ using a flow of nitrogen and diluted with 5 mL water.
  • the resulting mixture was loaded on a 20cc Oasis HLB cartridge (Waters), which was previously washed with acetonitrile (10 mL) and equilibrated with 0.1 % TFA (3 x 10 mL).
  • a mixture was prepared containing 30 nmol PNA from a stock solution in water ( ⁇ 2 mM), 150 nmol peptide from a stock solution in NMP ( ⁇ 10 mM), 0.2 ⁇ 2,6-lutidine (1.7 ⁇ ) and 1 ⁇ diisopropylethylamine (5.7 ⁇ ).
  • 7.5 ⁇ of 20 mM CuSCVTBTA solution (150 nmol) premixed in a 1 :1 mixture of DMSO and 10 ⁇ of a 20 mM solution of sodium ascorbate (200 nmol) were added to this mixture.
  • the mixture was left for one hour and quenched with 100 ⁇ 0.2 M EDTA 40% acetonitrile and 800 ⁇ TBS 40% acetonitrile.
  • the solution obtained was vortexed and loaded in two batches of 500 ⁇ on a streptavidin HP SpinTrap (GE Healthcare UK Limited) and each batch was incubated for 20 minutes while being mixed by inversion.
  • the column was washed with 400 ⁇ 0.1 M EDTA in TBS 40% acetonitrile and with 5x 400 ⁇ TBS 40% MeCN.
  • the conjugate was released from the resin by reaction for with 2x 20 minutes with 2x 400 ⁇ 10 mM TCEP in TBS 40% acetonitrile and the solid support washed with 200 ⁇ 40% acetonitrile in TBS.
  • the solutions collected were combined and freeze-dried.
  • the solid was dissolved in 500 ⁇ 20% acetonitrile and loaded on an equilibrated 1 cc Oasis HLB cartridge (Waters) together with 500 ⁇ 0.1 % TFA.
  • the column was washed with 3x 1 ml 0.1 % TFA, 3x 1 ml 5% acetonitrile 0.1 % TFA and 1x 1 ml 10% acetonitrile 0.1 % TFA. These extensive washes are required to remove the TCEP from the cartridge.
  • the conjugates are released using 500 ⁇ 60% acetonitrile and diluted with 50 ⁇ 0.1 % TFA.
  • the resulting solution was freeze-dried and dissolved in 500 ⁇ water. The concentration was determined and the solution was stored in the freezer for use in cellular assays. Splice-redirection assay
  • the splicing redirection assay was carried out similarly to a previously reported procedure with minor changes. 6 The assay was performed using 48-well plates with cells seeded the previous day (7.5x10 4 cells per well) and a total volume 100 ⁇ per well for the conjugates' solutions in OptiMEM. RT PGR experiments were carried out as reported in previous publications. 120 All experiments were performed in triplicates, unless specifically stated as designed one-point experiments.
  • the PNA contains a C-terminal S-trityl-protected Cys residue, which was introduced at the first step of solid-phase synthesis. After deprotection and cleavage from the solid support, a biotin group was introduced on to the N3-PNA705 by disulfide bridge formation via reaction of the C-terminal Cys residue with commercially available ⁇ /-[6- (biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (EZ-linkTM HPDP Biotin). This straight forward reaction provided the N 3 -PNA(705)-S-S-biotin after HPLC purification in 80% yield.
  • test library was narrowly based around a known relatively short (17 amino acids) CPP, Pip5h, which we have previously evaluated in exon skipping as a conjugate with a PMO targeting the pre-mRNA of dystrophin 10 , but which was not chosen for further development.
  • Peptide variants included modifications in the central 5-amino acid core (LB1_1 - 8) or on one of the flanking Arg-rich flanking regions (LB1_9 - 16) of the sequences. In each case an /V-terminal alkyne was introduced using 4-pentynoic acid through standard amide bond formation as the final coupling step (Scheme 2, Figure 8).
  • the PNA705 conjugates of LB1 were assessed for their ability to enter HeLa pLuc705 cells and redirect splicing, resulting in up-regulation of luciferase expression. Results were obtained with 48-well plates, which were used for all screening assays shown in this work. Several conjugates were purified by RP-HPLC and assayed, which provided similar results compared to the crude conjugates obtained by SELPEPCON ( Figure 22).
  • LB1-PNA705 conjugate library was assayed at a single-concentration (5 ⁇ ) and the results analysed as the fold increase in luminescence compared to buffer only ( Figure 2).
  • Several of the LB1-PNA705 constructs were strongly active, as was expected for Arg- rich peptides. Substantial reduction in the number of Arg residues (e.g. multiple replacements by His, Ser or Glu) led to significantly lower activity (LB1_10 - 12). When the Arg residues were replaced by Lys, retaining the overall positive charge, some splicing redirection activity was retained (LB1_9 and 13 - 16).
  • LB1-PNA705 conjugates differ slightly from CPP-PNA constructs previously synthesized by us. 6 ' 10 Thus LB1-PNA conjugates contain an additional free Cys residue and the peptide to PNA conjugation is N- to -terminus rather than C- to /V-terminal conjugation used in previous work.
  • LB1_1 containing the original Pip5h sequence showed similar splicing redirection activity to that of previously synthesised Pip5h-PNA705 (data not shown).
  • variations in splicing redirection activity observed in the HeLa cells were not identical to those that might have been expected based on knowledge of exon skipping effects in mouse mdx muscle cells for which the Pip5 and the later Pip6 series were designed.
  • 10 11 Scrambling of the hydrophobic core (LB1_3), replacement by Ala residues (LB1_4), substitution of Leu by Pro (LB1_8) or a negatively charged Glu (LB1_6) all appeared to be detrimental to the activity (Figure 2).
  • SELPEPCON can be effective for study and optimization of the amino acid sequence of a CPP for delivery of a cargo into a particular cell system and where a cellular assay is available.
  • peptide library LB2 consisting of 78 peptides that included many well-known CPPs, some sequences obtained from searching scientific literature, and some newly designed (Tables 3-5, Figures 12-14).
  • the peptides in this library consisted of both hydrophobic and hydrophilic peptides, ranged in length from 6-28 amino acids and carried a net charge ranging from -1 to +13.
  • the library consisted of standard L-amino acids with the addition of ⁇ -alanine (B) and ⁇ -aminocaproic acid (X), which are commonly found in CPPs. 3 ⁇ 4 c Since it was found that free thiol groups inhibited the copper-catalysed cycloaddition reaction, Cys residues could not be introduced using Cys(Trt), which is the standard protecting group used in peptide synthesis. Instead an alternative procedure was developed using S-te t-butylthio-L-cysteine, which contains a disulfide-protecting group that is stable to TFA cleavage but which is released in the reducing step of the conjugate workup procedure.
  • the LB2 peptides were conjugated to PNA705 using the procedure described previously for LB1 conjugations. Most conjugates were obtained in excellent yield (>60% average, Tables 3 to 8, Figures 12 to 17). As in the case of LB1 , a small amount of unconjugated PNA was observed by MALDI-TOF mass-spectroscopy and HPLC for all conjugates obtained ( Figures 20(D) - 21(B)). Out of the 72 peptides where there was a peptide product available for conjugation, 10 conjugates were either not obtained or showed only small amounts of conjugate product compared to unconjugated PNA following affinity purification and desalting.
  • the four LB2-PNA705 conjugates that contained Cys residues showed by mass-spectrometry impurities that resulted from a statistical mixture of te/f-butylthio (StBu) modifications. Some modification was even observed of the Cys residue that remains attached to PNA705 after release from the affinity support, which was evidenced by the fact that conjugate masses were found corresponding to two StBu groups for conjugates that contained only one Cys residue in the peptide part. This may be because residual free StBu that is not effectively removed by the OASIS cartridge workup of the conjugates can slowly allow StBu disulfides to form with available Cys residues in the conjugates.
  • StBu te/f-butylthio
  • StBu disulfide protecting groups are clearly cleaved off during the reduction step in SELPEPCON, since the alternative use of centrifugal filter units (Amicon Ultra 3K 0.5 ml, Milipore Ireland Ltd) to remove salts, TCEP reducing agent and StBu did provide the main product conjugates without StBu modifications. However, this procedure resulted in low yields, especially for hydrophobic conjugates.
  • the LB2-PNA705 conjugate library was screened at 5 ⁇ concentration for their ability to enter HeLa pLuc705 cells and redirect splicing, resulting in up-regulation of luciferase expression (Figure 3).
  • a significant number of active conjugates were identified from this initial screen, showing 20- to 200-fold increases in luminescence values.
  • high error-bars were observed for conjugates that showed over a 20-fold increase, because conjugates could not all be tested in a single 48-well plate experiment and samples with high luminescence readouts show large variability between different experiments.
  • the main purpose of the luciferase readout is to distinguish active from inactive conjugates.
  • a follow-up to screening can provide further information on selected candidate
  • LB2_7 contains the same Pip1 sequence as the positive control.
  • the PNA705 conjugate was N- to A erminally linked as obtained through SELPEPCON and showed high splice correction activity at both 2.5 and 5 ⁇ .
  • LB2_30 showed significantly lower luciferase activity.
  • the saturation of the splicing redirection observed at 5 ⁇ can explain the large variation in the luciferase activity assay.
  • these conjugates show the same activity trend by RT-PCR as seen in the luciferase readout obtained at 5 ⁇ , highlighting the difference in sensitivity between the two methods.
  • the SELPEPCON methodology described above has been exemplified by use of libraries of CPPs conjugated to a PNA705 cargo where a reliable cell assay is available to gauge the effectiveness of the peptides in delivering the cargo into the nucleus of HeLa cells.
  • the methodology is in principle applicable to any bio-cargo capable of being functionalized by an azido group, such as PMO or other oligonucleotide types, siRNA, peptides or other biomolecules where there is a need to search for peptides that when conjugated to a cargo may enhance its delivery into cells or for cell or tissue targeting.
  • the alkyne was placed on the C-terminus by use of A/-Fmoc- L-bishomopropargylglycine-OH (Fmoc- Bpg-OH, Chiralix, Nijmegen, The Netherlands) during solid-phase peptide synthesis. Both peptides were obtained in good yield.
  • the double glycine spacer of LB1_18 did not appear to be necessary to improve the conversion in the conjugation reaction, since both conjugates were readily obtained in excellent yield and purity.
  • the resultant libraries are also suitable for other screening purposes such as
  • GNGKKGTSFAEYWNLLSPC (solPMI-SH) was synthesized on solid support by solid phase synthesis and modified at the N-terminal amino group using 5-azido pentanoic acid.
  • the azide modified PMI (N 3 -solPMI-SH) was then modified with a biotin function using EZ-link HPDP-Biotin (Thermo Scientific).
  • the product (N3-solPMI-S-S-biotin) was purified by HPLC and used for conjugation reactions. Additionally, N3-solPMI-S-StBu was synthesized in order to perform several test click-conjugations in which the product was be isolated by HPLC-purification.
  • the obtained constructs were used in a cell-based assay.
  • a p53 reporter plasmid containing 14 copies of a p53 enhancer element sequence upstream of the luciferase gene was transfected into several cell-lines and the luciferase readout was determined using Nutlin-3, which is a small-molecule cell-permeable inhibitor of the MDM2-P53 interaction with a K d similar to that of PMI in vitro.
  • Nutlin-3 is a small-molecule cell-permeable inhibitor of the MDM2-P53 interaction with a K d similar to that of PMI in vitro.
  • a good response was found in MCF-7 cells, however it was found that most of the conjugates appeared to be toxic at concentrations of 5 ⁇ and above. Assaying the peptides at 1 and 2 ⁇ a small dose response was observed.
  • MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro
  • TBTA tris- (benzyltriazolylmethyl)amine
  • the solid support was as supplied by Intavis (Tentagel, 0.2 mmol g ⁇ 1 ). Double coupling steps were used with a PyBop/NMM coupling mixture followed by acetic anhydride capping after each coupling step.
  • the PMI sequence 23 containing a C-terminal C and an N-terminal G-N-G-K-K-G sequence for solubility was synthesised on solid support (see above).
  • Terminal azide functionalization involved a manual coupling procedure using 5-azido penanoic acid and standard coupling reagents.
  • the peptide was cleaved from the support and deprotected by addition of TFA (1.5 ml_) containing 5% triisopropylsilane/2.5% water and 1 % phenol with shaking for 2 hours. The mixture was filtered and concentrated.
  • the crude PNA was isolated by cold diethyl ether precipitation, dissolved in water, filtered and purified by HPLC.
  • a Phenomenex Semiprep C18 Jupiter column 250 x 10.00 mm, 5 micron was used with the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 20% B 2-25 min 20%-60% B 25-30 min 60%-90% B (retention time: 18.1 min).
  • the fractions containing the compound were combined and freeze-dried giving a fluffy white solid (yield 48% based on support loading). Mass, expected: m/z 2255 found: m/z 2253.
  • Alkyne modified CPP-peptides were taken from library LB2 used for the synthesis of PNA705 conjugates.
  • a mixture was prepared containing 30 nmol N3-solPMI-S-S-biotin from a stock solution in water ( ⁇ 2 mM), 150 nmol alkyne modified peptide from a stock solution in NMP ( ⁇ 10 mM), 0.2 ⁇ 2,6-lutidine (1.7 ⁇ ) and 1 ⁇ diisopropylethylamine (5.7 ⁇ ).
  • 7.5 ⁇ of 20 mM CuSCVTBTA solution (150 nmol) premixed in a 1 :1 mixture of DMSO and 10 ⁇ of a 20 mM solution of sodium ascorbate (200 nmol) were added to this mixture.
  • the mixture was left for one hour and quenched with 100 ⁇ 0.2 M EDTA 40% acetonitrile and 800 ⁇ TBS 40% acetonitrile.
  • the solution obtained was vortexed and loaded in two batches of 500 ⁇ on a streptavidin HP SpinTrap (GE Healthcare UK Limited) and each batch was incubated for 20 minutes while being mixed by inversion.
  • the column was washed with 400 ⁇ 0.1 M EDTA in TBS 40% acetonitrile and with 5x 400 ⁇ TBS 40% MeCN.
  • the conjugate was released from the resin by 2x 20 minutes reactions with 2x 400 ⁇ 10 mM TCEP in TBS 40% acetonitrile and the solid support washed with 200 ⁇ 40% acetonitrile in TBS.
  • the solutions collected were combined and freeze-dried.
  • the solid was dissolved in 500 ⁇ 20% acetonitrile and loaded on an equilibrated 1 cc Oasis HLB cartridge
  • the assay is carried out in 48-well plated MCF-7 cells at 50-60 % confluency. First, 250 mg per well of p53-Luc plasmid is transfected by lipofection with Lipofectamine2000. Then the next day cells are incubated for 4 h with CPP-PMI conjugates in OptiMEM, followed by addition of standard media of DMEM/10% FBS and further incubation for 20 h. Next day media is changed for the fresh DMEM/105FBS and cells are left for another 24h incubation followed by Luciferase assay according to standard protocol.
  • the biotinylated LB2_9_SH was conjugated to the 3'-end of the PMO through its C- terminal carboxyl group by reaction with HBTU/HOAt/DIPEA in NMP for 2 h at room temperature.
  • the resulting solution was immobilized on a streptavidin HP SpinTrap column and the conjugate was released by reaction with TCEP.
  • the resulting solution was purified on an Oasis HLB cartridge to yield the desired conjugate.
  • the free thiol group in the conjugate was capped by treatment with iodoacetamide in sodium bicarbonate solution for 2 h in the dark and excess iodoacetamide was removed through the use of an Amicon® Ultra-1 ml_ centrifugal filter unit as before.
  • MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro
  • BioSpectrometry workstation A stock solution of 10 mg mL 1 of sinapinic acid in 60% acetonitrile in water was used as matrix. Error bars are ⁇ 0.1 %. Reversed phase HPLC purifications and analyses were carried out using a Varian 940-LC. A Nanodrop 2000 UV analyser (Thermo Scientific) was used for the quantification of PMO concentrations.
  • Bifunctional PMO (3'-disulfide, 5' primary amine) as well as unmodified PMO were purchased from Gene Tools LCC, Philomath, OR, USA.
  • the PMO sequence is 5' to 3' GGCCAAACCTCGGCTTACCTGAAAT.
  • Optical Density was measured at 265 nm using a molar absorption coefficient of 259.21 cnr 1 M ⁇ 1 as provided by the supplier.
  • EZ-link HPDP Biotin was bought from Thermo Scientific, Waltham, MA, USA.
  • the PMO (3'-disulfide, 5' primary amine) was dissolved in sterile water (around 1 nmol/ ⁇ , 1 mM). The concentration of the solution and the amount of PMO was determined and 1/5 th of the same volume of 10 mM TCEP in TBS was added, and the solution mixed for 30 min. If there are any particles, the solution was filtered using a SPIN-X column. The solution was loaded in an Amicon ® Ultra (Merck, Millipore, Darmstadt, Germany) 0.5 ml centrifugal filter and the solution concentrated and washed several times (5x) with water. The washed HS-S-PMO-NH2 was transferred to a new tube and lyophilized. Mass, expected: m/z 8780 found: m/z 8788.
  • LB2_9 peptide (Table 3, Figure 12) was synthesized N-terminally acetylated and with an additional three-amino acid CXB C-terminal extension where the C-terminus is a free carboxylic acid (Ac-RXRRBRRXRYQFLI RXRBRXRCXB where B is ⁇ -alanine and X is aminohexanoic acid) on an Fmoc-p-Ala-Wang resin ( 00-200 mesh, 0.6 mmol/g) using Fmoc chemistry on a CEM LibertyTM microwave peptide synthesizer (Buckingham, UK).
  • the side chain protecting groups used were trifluoroacetic acid labile.
  • the peptide was synthesized on a 0.1 mmol scale using a 5-fold excess of Fmoc-protected amino acids (0.5 mmol), which were activated using PyBOP (5-fold excess) in the presence of DIPEA.
  • Piperidine (20% v/v in DMF) was used to remove N-Fmoc protecting groups. The coupling was carried out once at 75 °C for 5 min at 60-watt microwave power except for arginine residues, which were coupled twice each. Each deprotection reaction was carried out at 75 °C twice, once for 30 sec and then for 3 min at 35-watt microwave power.
  • the resin was washed with DMF (3 x 50 ml_) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA.
  • the peptide was cleaved from the solid support by treatment with a cleavage cocktail consisting of trifluoroacetic acid (TFA): 3,6-dioxa-1 ,8-octanedithiol (DODT): H 2 0: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1 %, 10 mL) for 3 h at room temperature. Excess TFA was removed by blowing N 2 through the peptide solution.
  • TFA trifluoroacetic acid
  • DODT 3,6-dioxa-1 ,8-octanedithiol
  • TIPS triisopropylsilane
  • the cleaved peptide was precipitated via the addition of ice-cold diethyl ether and centrifuged at 3000 rpm for 2 min. The peptide pellet was washed in ice-cold diethyl ether thrice. The crude peptide was dissolved in water, analyzed and purified by RP-HPLC on Phenomenex Jupiter columns (4.6 x 250 mm, C18, 5 ⁇ ) and (21.2 X 250 MM, C18, 10 ⁇ - ⁇ ) respectively.
  • a Phenomenex Jupiter column (21.2 ⁇ 250 mm, C18, 10 mm) was used at a flow rate 10 mL/min with the following gradient (A: 0.1 % TFA, B: 90% CH 3 CN, 0.1 % TFA) 0-30 min 10%-70% B 30-33 min 70%-90% B.
  • the fractions containing the desired peptide were combined and freeze dried to yield the biotin-modified peptide as a white solid (48% yield). Mass, expected: m/z 3588 found: m/z 3595.
  • biotinylated LB2_9-SH was conjugated to the 3'-end of the 25-mer PMO antisense sequence targeting mouse dystrophin exon-23, through its C-terminal carboxyl group.
  • the conjugate was released from the resin by 2 ⁇ 20 min reaction with TCEP (2 400 mL of 10 mM in 20% CH 3 CN TBS) and the resin washed with TBS (200 mL). The resulting solutions were combined and lyophilized. The resultant white solid was dissolved in 0.1 % TFA 10% CH 3 CN (500 mL) and loaded on to an equilibrated 1 cc Oasis HLB cartridge (Waters) together with 0.1% TFA (500 mL). The column was washed with 0.1 % TFA (3 ⁇ 1 mL), 5% CH 3 CN in 0.1 % TFA (3 1 mL) and 10% CH 3 CN in 0.1 % TFA (1 1 mL).
  • the conjugate was released from the cartridge via the addition of 60% CH 3 CN in 0.1 % TFA (500 mL). The resulting solution was lyophilized to yield the desired conjugate as a white solid (42 % yield). Mass, expected: m/z 1 1564 found: m/z 1 1583.
  • the conjugate (10 nmol) was dissolved in sodium bicarbonate solution (25 mL of 0.1 M, pH 8.0) and iodoacetamide (23 mg, 250 nmol) was added. The solution was left for 2 h in the dark and diluted with water (975 mL). Excess iodoacetamide was removed through the use of an Amicon® Ultra-1 mL centrifugal filter unit with a 3000 molecular weight cut off. The resulting filtrate was lyophilized to yield the desired S-capped product (55%). Mass, expected: m/z 1 1622 found: m/z 1 1642.

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Abstract

A method is disclosed for the rapid synthesis and isolation of peptide-cargo conjugates in which a peptide is chemically conjugated to a cargo molecule.

Description

Peptide Conjugates
Field of the Invention
The present invention relates to peptide conjugates.
Background to the Invention
Efficient cell delivery of bio-molecules, such as oligonucleotides and peptides, is a major hurdle in development of novel therapeutics. As a result, higher drug dosages are often required than would otherwise be needed, which increases costs and the possibility of off target effects. A promising method of enhancing cell delivery is by use of cell-penetrating peptides (CPPs) to facilitate cell-uptake.1 CPPs often contain positive charges and/or hydrophobic elements. Some are based on cell-permeable peptides obtained from larger proteins and are known as protein transduction domains (PTD), whereas others are synthetically derived. CPPs have been shown to deliver a range of different biologically active cargoes into cells and in vivo, including proteins, peptides, oligonucleotide analogues, siRNAs and small molecule drugs.2
In the case of oligonucleotide cargoes, CPPs can either be complexed with or conjugated covalently to the cargo. For example, CPP conjugates of charge neutral antisense phosphorodiamidate morpholino oligonucleotides (PMO) or peptide nucleic acids (PNA) have been shown to be very effective in induction of splicing redirection or exon skipping in cells and in vivo? However, there have been very many CPPs proposed, and individual research groups often utilise their own preferred peptide sequences. No single CPP has been found to be universally successful for the conjugate delivery of all cargoes into all cell types. Instead for a new drug target or application, success is often only achieved through painstaking conjugate synthesis on an individual basis and search for a suitable peptide sequence by trial and error using a cell or in vivo assay. Only a small number of CPPs (commonly 2 or 3 known CPPs) can usually be tested as bio-cargo conjugates because of the difficulty and the time-consuming nature of their syntheses, even though cell-screening reporter assays, e.g. luciferase-based, are frequently available and adaptable for high-throughput. Summary of the Invention
The inventors have now provided a method for the rapid synthesis, and isolation of peptide-cargo conjugates in which a peptide is chemically conjugated to a cargo molecule. This advance in the art makes possible the rapid and high throughput screening of peptide-cargo conjugates for the first time.
In a first aspect of the present invention a method of forming a peptide-cargo conjugate is provided, the conjugate comprising a peptide bonded to a cargo molecule, the method comprising:
(i) contacting a peptide attached to a first functional group at the N- or C- terminus with a cargo construct comprising a cargo molecule attached to a second functional group, the cargo molecule also comprising a tag, under conditions that allow reaction between the functional groups to form a bond between the peptide and cargo molecule, thereby forming a peptide- cargo construct conjugate in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(ii) partitioning the peptide-cargo construct conjugate from unreacted peptide by binding the tag to a capture element;
(iii) releasing the peptide-cargo conjugate from the tag.
The method may optionally comprise a washing step after step (i) and/or step (ii).
In a second aspect of the present invention a method of forming peptide-cargo conjugates is provided, the conjugates each comprising a peptide bonded to a cargo molecule, the method comprising:
(a) preparing a plurality of peptides each attached to a first functional group at the N- or C- terminus;
(b) preparing a plurality of cargo constructs each comprising a cargo molecule
attached to a second functional group, the cargo molecule also comprising a tag;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide; (d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags.
The method may optionally comprise a washing step after step (c) and/or step (d). In preferred embodiments each of the plurality of cargo constructs in (b) have the same cargo molecule, and/or the same functional group and/or tag.
In another aspect of the present invention a method of forming a peptide-cargo conjugate, the conjugate comprising a peptide bonded to a cargo molecule, is provided, the method comprising:
(i) contacting a peptide attached to a first functional group at the N- or C- terminus with a cargo construct comprising a cargo molecule attached to a second functional group, the peptide molecule also comprising a tag, under conditions that allow reaction between the functional groups to form a bond between the peptide and cargo molecule, thereby forming a peptide-cargo construct conjugate in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(ii) partitioning the peptide-cargo construct conjugate from unreacted peptide by binding the tag to a capture element;
(iii) releasing the peptide-cargo conjugate from the tag.
In another aspect of the present invention a method of forming peptide-cargo conjugates, the conjugates each comprising a peptide bonded to a cargo molecule, is provided, the method comprising:
(a) preparing a plurality of peptides each attached to a first functional group at the N- or C- terminus, the peptide molecule also comprising a tag;
(b) preparing a plurality of cargo constructs each comprising a cargo molecule
attached to a second functional group;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide; (d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags.
The method may optionally comprise a washing step after step (c) and/or step (d). In preferred embodiments each of the plurality of cargo constructs in (b) have the same cargo molecule, and/or the same functional group and/or tag.
In another aspect of the present invention a method of generating a library of peptide- cargo conjugates is provided, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus; (b) preparing a plurality of cargo constructs of the same type, each comprising a cargo molecule attached to a second functional group, the cargo molecule also comprising a tag;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element; (e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
The method may optionally comprise a washing step after step (c) and/or step (d).
In a further aspect of the present invention there is provided a method of screening a plurality of peptide-cargo conjugates for a functional property of the conjugate, the method comprising simultaneously assaying a plurality of different peptide-cargo conjugates for said functional property.
In another aspect of the present invention a method of generating a library of peptide- cargo conjugates, the library having a plurality of different peptide-cargo conjugates, is provided, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus, the peptide molecule also comprising a tag;
(b) preparing a plurality of cargo constructs of the same type, each comprising a
cargo molecule attached to a second functional group;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
The method may optionally comprise a washing step after step (c) and/or step (d).
In a further aspect of the present invention there is provided a method of screening a plurality of peptide-cargo conjugates for a functional property of the conjugate, the method comprising simultaneously assaying a plurality of different peptide-cargo conjugates for said functional property.
The assays of respective different peptide-cargo conjugates may be performed in parallel. In some embodiments of the method, screening of the peptide-cargo conjugates comprises use of the same cargo molecule together with the use of different peptides. In some embodiments of the method, screening of the peptide-cargo conjugates comprises use of different peptides together with different cargo molecules.
The method of screening may further comprise providing a library of different peptide- cargo conjugates on which to perform the assay. As such, the method of screening may further comprise generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus;
(b) preparing a plurality of cargo constructs of the same type, each comprising a cargo molecule attached to a second functional group, the cargo molecule also comprising a tag; (c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide; (d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
Alternatively, the method of screening may further comprise generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus, the peptide molecule also comprising a tag; (b) preparing a plurality of cargo constructs of the same type, each comprising a cargo molecule attached to a second functional group;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element; (e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
In a further aspect of the present invention a library of peptide-cargo conjugates is provided, the library comprising a plurality of containers each containing a quantity of an isolated or substantially purified peptide-cargo conjugate. Each peptide-cargo conjugate is preferably different with respect to either the peptide or cargo component.
In another aspect of the present invention a peptide-cargo construct conjugate comprising a peptide bonded to a cargo construct comprising a cargo molecule and a releasable tag is provided, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide. The peptide-cargo construct conjugate is preferably provided in isolated form. In a further aspect of the present invention a plurality of peptide-cargo construct conjugates immobilised on a solid support through association of a tag with a capture element is provided, wherein each peptide-cargo construct conjugate comprises a peptide bonded to a cargo construct comprising a cargo molecule and a tag capable of association with a capture element on the solid support, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide molecule.
In embodiments of the library of peptide-cargo conjugates, the peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates the cargo construct may comprise a cleavable linker attached to the tag and to the cargo molecule, the cleavable linker linking the tag and cargo molecule, preferably whereupon cleavage at (or of) the linker releases the peptide-cargo conjugate molecule from the tag.
In another aspect of the present invention a peptide-cargo construct conjugate is provided, the peptide-cargo construct conjugate comprising a peptide having a releasable tag, the peptide bonded to a cargo construct comprising a cargo molecule, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide.
In a further aspect of the present invention a plurality of peptide-cargo construct conjugates immobilised on a solid support through association of a tag with a capture element is provided, wherein each peptide-cargo construct conjugate comprises a peptide having a tag capable of association with a capture element on the solid support, the peptide bonded to a cargo construct comprising a cargo molecule, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide molecule.
In embodiments of the library of peptide-cargo conjugates, the peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates the peptide may comprise a cleavable linker attached to the tag and to the peptide, the cleavable linker linking the tag and peptide, preferably whereupon cleavage at (or of) the linker releases the peptide- cargo conjugate molecule from the tag.
In embodiments of the present invention the peptide may have a maximum length of 60 amino acids and a minimum length of 2 amino acids. In some embodiments the peptide has a length selected from the group consisting of: 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 29 amino acids, 5 to 28 amino acids, 5 to 27 amino acids, 5 to 26 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 22 amino acids, 5 to 21 amino acids, or 5 to 20 amino acids.
In preferred embodiments the conjugates comprise a peptide covalently bonded to the cargo molecule.
In preferred embodiments the first and second functional groups form a pair having selective reactivity towards each other. In some embodiments the first and second functional groups are selected from one of: an azide and an alkyne; a thiol and an alkene; a thiol and an alkyne; a diene and a dienophile; an isonitrile and a tetrazine; an epoxy and an aziridine; an amine and an isocyanate, an active methylene and an activated olefin; a thioester and a thiol (e.g. cysteine thiol) [native ligation]; an aldehyde and a hydrazine [hydrazide]; an aldehyde and an aminoxy derivative [oxime]. In some embodiments, one of the functional groups may need to be activated by prior reaction, for example where the first functional group is an amine and the second functional group is a carboxylic acid that is activated by reaction with a coupling agent such as HBTU in the presence of HOAt.
In some embodiments the first functional group is attached to the terminal amine of the peptide.
In preferred embodiments the cargo molecule is an oligonucleotide, an oligonucleotide analogue, a peptide or a peptide analogue. In some embodiments the cargo molecule is a peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), or siRNA.
Description
A method of peptide conjugate library synthesis is needed that is quick and efficient and is suitable for subsequent use of rapid screening assays.4 Following cell assay of an initial library of conjugates using peptides of widely ranging sequence, fine-tuning of a peptide candidate can then be accomplished either by further narrower library synthesis and cell re-assay, or by more conventional synthesis if further screening is to be carried out by an in vivo assay. The most time-limiting step in synthesis of peptide-cargo conjugates is generally not the conjugation itself but the individual purifications required for peptide, cargo and conjugate. Recently, parallel multi-peptide synthesis machines have become available as well as new methods for rapid affinity-based purification of bio-molecules (e.g. histidine tags or biotin-streptavidin)4b that provide an opportunity both for rapid synthesis and for reaction workup and purification. In addition, recent developments in bio-orthogonal "click"-type ligation reactions provide a chemical basis for efficient conjugation of bio-molecules in aqueous solution.5
These new methodologies inspired us to develop a new strategy for the SELection of PEPtide CONjugates (SELPEPCON) of bio-cargoes that can act as an initial parallel synthesis approach useful for therapeutic screening. SELPEPCON allows for convenient and rapid parallel conjugation reactions and workup that avoid the need for HPLC purification (Figure 1 ). The method utilizes a functionalized cargo linked through a cleavable linker to an affinity tag. The functionalized cargo is conjugated to the peptide after which it is purified by immobilization and isolated by release of the tag. Affinity purification is very rapid and is readily automated, making the overall strategy very suitable for high-throughput synthesis of conjugates for screening.
In this specification "peptide" has its normally meaning in the art. The term "peptide" includes a molecule comprising a contiguous sequence of amino acid residues where adjacent amino acids are joined by a peptide bond. As such, where the peptide is linear it normally has an N-terminus and a C-terminus, which are available for modification.
The peptide may contain amino acids of any type and in any sequence. Amino acids may be natural or non-natural amino acids, D- or L- amino acids and may be modified amino acids. Examples of non-natural amino acids include: 6-aminohexanoic acid (also called aminocaproic acid or ε-aminocaproic acid or Ahx), 4-aminobutyric acid, aminocaprylic acid, β-alanine, p-aminobenzoic acid and isonipecotic acid. Peptides according to the present invention may have a maximum length of 60 amino acid residues, or one of 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , or 10 residues. Peptides according to the present invention may have a minimum length of 2 amino acid residues, or one of 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues. As such, peptides may have a length in the range of one of 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 29 amino acids, 5 to 28 amino acids, 5 to 27 amino acids, 5 to 26 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 22 amino acids, 5 to 21 amino acids, 5 to 20 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 29 amino acids, 6 to 28 amino acids, 6 to 27 amino acids, 6 to 26 amino acids, 6 to 25 amino acids, 6 to 24 amino acids, 6 to 23 amino acids, 6 to 22 amino acids, 6 to 21 amino acids, or 6 to 20 amino acids, Preferably, consideration of the length of the peptide does not include the length of the cargo molecule.
The peptide may be any kind of peptide. Preferably it is a linear peptide. In some embodiments it is a cell-penetrating peptide (CPP), or is a candidate cell-penetrating peptide. The cargo molecule may be any small molecule, e.g. small molecule drug, peptide, cyclic peptide, protein, pharmaceutical or therapeutic (e.g. molecular weight less than one of 20,000 Da, 15,000 Da, 10,000 Da, 5,000 Da, 3000 Da or 1000 Da). The cargo molecule may be a nucleic acid, antisense oligonucleotide, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA)), or siRNA.
In some preferred embodiments the cargo molecule is an oligonucleotide, an
oligonucleotide analogue, a peptide or a peptide analogue. Preferred oligonucleotide or peptide cargoes (or their analogues) are linear molecules, such as peptides,
oligonucleotides or their analogues. Oligonucleotides may be single stranded or double stranded. Suitable oligonucleotide cargo molecules include conventional DNA and RNA
oligonucleotides, which may include modified nucleotides, such as 2'modification of ribose, e.g. 2 -O-methyl (2'-OMe), 2'-amino (2'-NH) or 2'-Fluoro (2'-F) and/or 8-position purine modifications and/or 5-position pyrimidine modifications), LNA, siRNA. Suitable oligonucleotide analogues include PNA and PMO.
Where the cargo molecule is an oligonucleotide or oligonucleotide analogue it may have a maximum length of 5000 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs). The maximum length may be one of 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs). It may have a minimum length of 5 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs). The minimum length may be one of 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides (or nucleotide analogues or bases or [for a double stranded molecule] base pairs).
In this specification a "peptide-cargo conjugate" refers to a molecule in which a peptide, as described herein, is bonded (preferably covalently) to a cargo molecule, as described herein. The covalent bond is preferably formed between the N- or C- terminus of the peptide and the cargo molecule. In this specification the starting materials for reaction include a peptide attached to a first functional group and cargo molecule attached to a second functional group. The attachment is preferably a covalent or ionic bond, most preferably a covalent bond. The first and second functional groups form a pair having reactivity, preferably selective reactivity, towards each other such that upon contact a chemical reaction occurs leading to the formation of a covalent or ionic bond linking the peptide and cargo molecule. In some instances the reaction may be subject to catalysis.
A range of functional groups are available, including pairs of functional groups that are bio-orthogonal, having selective reactivity towards each other.
In particular, the first and second functional groups may form a pair of functional groups having reactivity using Click chemistry. Click chemistry is preferred because typical Click chemistry reactions are simple, require simple or no purification and give high yields. Click chemistry reactions include cycloadditions and additions to double or triple bonds. More specific examples include [3+2] cycloadditions, such as the Cu(l)-catalyzed stepwise Huisgen 1 ,3-dipolar cycloaddition, thiol-alkene and thiol alkyne click reactions, Diels-Alder reactions and inverse electron demand Diels-Alder reactions,
[4+1] cycloadditions, nucleophilic substitution especially to small strained rings, non-aldol type carbonyl reactions, such as the formation of ureas, and Michael Additions. Other examples include native chemical ligation of a thiolate group of an N-terminal cysteine with a C-terminal thioester, hydrazide formation by reaction of an aldehyde with a hydrazine, or oxime formation by reaction of an aldehyde with an aminooxy derivative, e.g. a hydroxylamine.
Accordingly, suitable first and second functional group pairs include: an azide and an alkyne; a thiol and an alkene; a thiol and an alkyne; a diene and a dienophile; an isonitrile and a tetrazine; an epoxy and an aziridine; an amine and an isocyanate, an active methylene and an activated olefin, a thioester and a thiol; an aldehyde and a hydrazine; an aldehyde and an aminoxy derivative. Examples of each functional group suitable for the Click chemistry reactions are known in the art.
A particularly preferred functional group pair is an azide and an alkyne. Azides and alkynes may be used in the Huisgen 1 ,3-dipolar cycloaddition. The reaction forms a 1 ,2,3-triazole link. The alkyne may be a terminal or internal alkyne. The reaction of the azide with the alkyne may be catalysed. For example, the reaction may be a Copper(l)- catalyzed azide-alkyne cycloaddition (CuAAC), a Ruthenium-catalysed 1 ,3-dipolar azide- alkyne cycloaddition (RuAAC) or a Silver(l)-catalyzed azide-alkyne cycloaddition
(Ag-AAC). Alternatively, the reaction may be uncatalysed, e.g. where the alkyne is cylooctyne. The reaction conditions for such reactions are known in the art.
The choice of which functional group is the first functional group on the peptide or the second functional group on the cargo molecule is not particularly limited. The choice may be determined, for example, by ease of preparation of the peptide and cargo molecule and/or the compatibility of the functional group with other functional groups in the peptide or cargo molecule. For example, an isocyanate may not be suitable as the first functional group because of potential inter- or intra-molecular reaction with peptide amine groups. In preferred embodiments, the first functional group is an alkyne and the second functional group is an azide. When the first functional group is an alkyne and the second functional group is an azide, the method of the present invention preferably includes a Copper(l)-catalyzed azide-alkyne cycloaddition (CuAAC) to link the peptide to the cargo molecule. In these embodiments, the peptide-cargo conjugate contains a 1 ,2,3-triazole link. The alkyne functionality is preferably located on the peptide and the azide functionality on the cargo. The functional group may be attached to the peptide at any suitable position, but is preferably attached to the peptide at the N-terminus (preferably via bonding to the nitrogen of the terminal amine) or C-terminus (preferably via bonding to the carbon of the terminal carboxyl), and most preferably at the N-terminus via bonding to the nitrogen of the terminal amine. The functional groups may be incorporated in the peptide and/or cargo molecules during synthesis, e.g. as part of the final coupling step in peptide or oligonucleotide synthesis.
Attachment of the functional group to the cargo molecule may be at any suitable position. Where the cargo molecule is a linear molecule, attachment is preferably at one end of the molecule.
Where the cargo molecule is a peptide, attachment may be at the N- or C- terminus, as described above. In some embodiments N-terminal attachment is preferred. This may provide for a conjugate in which the N-terminus of the peptide is conjugated to the N- terminus of the cargo peptide. In other embodiments a conjugate is formed in which the N-terminus of the peptide is conjugated to the C-terminus of the cargo peptide. In yet other embodiments a conjugate is formed in which the C-terminus of the peptide is conjugated to the C-terminus of the cargo peptide.
Where the cargo molecule is an oligonucleotide or oligonucleotide analogue the functional group may be attached at either the 3' or 5' end.
In some embodiments attachment of the functional group may involve modification of the peptide or cargo to facilitate attachment of the functional group. In some embodiments the cargo molecule (or optionally the peptide) comprises a tag which is preferably attached to the cargo molecule or part of the cargo construct by a covalent or ionic bond. The tag is preferably releasable from the cargo molecule.
In some embodiments the cargo molecule (or optionally the peptide) is also attached to a cleavable linker. The cleavable linker is preferably attached to the tag and to the cargo molecule (or optionally the peptide), and may be positioned between the tag and cargo molecule (or optionally the peptide). Attachment of the cleavable linker to the tag and/or cargo molecule (or optionally the peptide) is preferably by covalent and/or ionic bond. As such, in some embodiments the cargo construct comprises a linear molecule having a tag at one end attached to a cleavable linker which is attached to the cargo molecule (which may also be a linear molecule). As such, the cleavable linker may be positioned between the tag and cargo molecule. The cargo construct may optionally have a linear arrangement of tag-cleavable linker-cargo molecule, in such an arrangement the cleavable linker being called an in-line cleavable linker. Cleavage of the cleavable linker leads to detachment/release of the tag (and optionally all or part of the cleavable linker) from the cargo construct.
Cleavable linkers are available that permit selective and controlled cleavage by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents [Leriche et al (Cleavable linkers in chemical biology. Bioorg Med Chem. 2012 Jan 15;20(2):571-82)].
In some embodiments, peptide-cargo construct conjugates are partitioned by capturing or immobilizing on a solid support using a tag incorporated into the cargo construct. For example, if the tag is biotin, beads having a capture element such as avidin, streptavidin, neutravidin, or extravidin, can be used to capture the peptide-cargo construct conjugates. The beads maybe washed to remove any free peptide.
A tag refers to a component that provides a means for attaching or immobilizing a cargo construct (and any peptide conjugated to it) to a solid support. A tag is a component that is capable of associating with a capture element.
A tag can be attached to or included in the cargo construct by any suitable method.
Generally, the tag allows the cargo construct to associate, either directly or indirectly, with a capture element that is attached to a solid support.
The capture element is typically chosen (or designed) to be highly specific in its interaction with the tag and to retain that association during subsequent processing steps or procedures. A tag can enable the localization of a cargo construct (or peptide-cargo construct conjugate) to a spatially defined address on a solid support.
Different tags can enable the localization of different cargo constructs to different spatially defined addresses on a solid support. A tag can be a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, biotin, polyhistidine, or any fragment or derivative of these structures, any combination of the foregoing, or any other structure with which a capture element can be designed or configured to bind or otherwise associate with specificity.
Generally, a tag is configured such that it does not interact intra-molecularly with either itself or the cargo construct to which it is attached or of which it is a part.
In one embodiment, the tag is biotin group and the capture element is a biotin binding protein such as avidin, streptavidin, neutravidin, or Extravidin. This combination may be conveniently used in various embodiments, as biotin is easily incorporated into cargo constructs during synthesis and streptavidin beads are readily available.
In one embodiment, the tag is polyhistidine and the capture element is nitrilotriacetic acid (NTA) chelated with a metal ion such as nickel, cobalt, iron, or any other metal ion able to form a coordination compound with poly-histidine when chelated with NTA. In one embodiment, a tag can associate directly with a probe on the surface of the solid support and covalently bind to the probe, thereby covalently linking the cargo construct to the surface of the solid support. In this embodiment, the tag and the probe can include suitable reactive groups that, upon association of the tag with the probe, are sufficiently proximate to each other to undergo a chemical reaction that produces a covalent bond. The reaction may occur spontaneously or may require activation, such as, for example, photo-activation or chemical activation, for example in one embodiment where the first functional group is an amine and the second functional group is a carboxylic acid that is activated by reaction with a coupling agent such as HBTU in the presence of HOAt so that an amide bond is formed. In one embodiment, the tag includes a diene moiety and the probe includes a dienophile, and covalent bond formation results from a spontaneous Diels-Alder conjugation reaction of the diene and dienophile. Any appropriate
complementary chemistry can be used, such as, for example, N-Mannich reaction, disulfide formation, native ligation, hydrazide formation, oxime formation, Curtius reaction, Aldol condensation, Schiff base formation, and Michael addition. Where a disulfide bond is the cleavable linker the reaction between tag and probe preferably does not rely on disulfide bond formation.
In some embodiments, the tag component is bi-functional in that it includes functionality for specific interaction with a capture element on a solid support and functionality for dissociating the cargo molecule to which it is attached from the tag. The means for dissociating the tag includes chemical means, photochemical means or other means depending upon the particular tag that is employed.
A capture element refers to a molecule that is configured to associate, either directly or indirectly, with a tag. A capture element is a molecule or type of multi-molecular structure that is capable of immobilizing the cargo molecule to which the tag is attached to a solid support by associating, either directly or indirectly, with the tag.
A capture element can be a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affybody, an antibody mimic, a cell receptor, a ligand, a lipid, bitoin, polyhistidine, or any fragment or derivative of these structures, any combination of the foregoing, or any other structure with which a tag can be designed or configured to bind or otherwise associate with specificity. A capture element can be attached to a solid support either covalently or non-covalently by any suitable method.
Due to the reciprocal nature of the interaction between a particular tag and capture element pair, a tag in one embodiment may be used as a capture element in another embodiment, and a capture element in one embodiment may be used as a tag in another embodiment. For example, a cargo construct with a biotin tag may be captured with streptavidin attached to a solid support in one embodiment, while a cargo construct with a streptavidin tag may be captured with biotin attached to a solid support in another embodiment.
In some embodiments, it is desirable to immobilize peptide-cargo construct conjugates to a solid support to enable the isolation of peptide-cargo construct conjugates and remove free peptide. As used herein, a releasable or cleavable element, moiety, or linker refers to a molecular structure that can be broken to produce two separate components. A releasable (or cleavable) element may comprise a single molecule in which a chemical bond can be broken, or it may comprise two or more molecules in which a non-covalent interaction can be broken or disrupted.
In some embodiments, it is necessary to spatially separate certain chemical groups or moieties from others in order to prevent interference with the individual functionalities or to provide for the attachment of a functional group. For example, in order to attach a functional group at the 3' or 5' end of an oligonucleotide cargo molecule. Spacing linkers may be used for this purpose.
Spacing linkers may be introduced into an oligonucleotide, PNA, PMO or peptide cargo molecules during synthesis and so can be comprised of number of phosphoramidite spacers, including but not limited to aliphatic carbon chains of length 3, 6, 9, 12 and 18 carbon atoms, polyethylene glycol chains of length 1 , 3, and 9 ethylene glycol units, or a tetrahydrofuran moiety (termed dSpacer (Glen Research) or any combination of the foregoing or any other structure or chemical component that can be designed or configured to add length along a phosphodiester backbone. In another embodiment, the spacing linker includes polynucleotides, such as poly dT, dA, dG, or dC or poly U, A, G, or C or any combination of the foregoing. In another embodiment, spacers include one or more abasic ribose or deoxyribose moieties. Spacing linkers may also be included at one or both ends of a cargo molecule, including oligonucleotide or peptide cargo molecules. Suitable spacing molecules include those described above. For peptide cargoes, spacing molecules may also include natural or non-natural amino acids, D- or L- amino acids or ethylene glycol or polyethylene glycol spacers. Examples of suitable non-natural amino acids include: 6-aminohexanoic acid, 4-aminobutyric acid, aminocaprylic acid, β-alanine, p-aminobenzoic acid and isonipecotic acid.
An in-line cleavable linker refers to a group of atoms that contains a releasable or cleavable element. In some embodiments, an in-line cleavable linker is used to join a cargo molecule to a tag, thereby forming a releasable tag.
An in-line cleavable linker may be chemically cleavable in that it includes a bond that can be cleaved by treating it with an appropriate chemical or enzymatic reagent. One example is a disulphide bond that can be cleaved by treating it with a reducing agent to disrupt the bond.
An in-line cleavable linker may be photo-cleavable (in that it includes a bond that can be cleaved by irradiating the releasable element at the appropriate wavelength of light). A solid support refers to any substrate having a surface to which molecules may be attached, directly or indirectly, through either covalent or non-covalent bonds. The solid support may include any substrate material that is capable of providing physical support for the capture elements or probes that are attached to the surface. The material is generally capable of enduring conditions related to the attachment of the capture elements or probes to the surface and any subsequent treatment, handling, or processing encountered during use.
The materials may be naturally occurring, synthetic, or a modification of a naturally occurring material. Suitable solid support materials may include silicon, graphite, mirrored surfaces, laminates, ceramics, plastics (including polymers such as, e.g., polyvinyl chloride), cyclo-olefin copolymers, agarose gels, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene ), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon, polyvinyl butyrate)), germanium, gallium arsenide, gold, silver. Additional rigid materials may be considered, such as glass, which includes silica and further includes, for example, glass that is available as Bioglass. Other materials that may be employed include porous materials, such as, for example, controlled pore glass beads, crosslinked beaded sepharose or agarose resins, or copolymers of crosslined
bisacrylamide and azalactone. Any other materials known in the art that are capable of having one or more functional groups, such as any of an amino, carboxyl, thiol, or hydroxyl functional group, for example, incorporated on its surface, are also
contemplated.
The material used for a solid support may take any of a variety of configurations ranging from simple to complex. The solid support can have any one of a number of shapes, including a strip, plate, disk, rod, particle, bead, tube, well, or column. The solid support may be porous or non-porous, magnetic, paramagnetic, or non-magnetic, polydisperse or monodisperse, hydrophilic or hydrophobic. The solid support may also be in the form of a gel or slurry of closely packed (as in a column matrix) or loosely-packed particles.
The solid support with attached capture element may be used to capture tagged peptide- cargo construct conjugates. By way of example, when the tag is a biotin moiety, the solid support could be a streptavidin-coated bead or resin such as Dynabeads M-280
Streptavidin, Dynabeads MyOne Streptavidin, Dynabeads M-270 Streptavidin
(Invitrogen), Streptavidin Agarose Resin (Pierce), Streptavidin Ultralink Resin,
MagnaBind Streptavidin Beads (Thermo Scientific), BioMag Streptavidin, Pro Mag Streptavidin, Silica Streptavidin (Bangs Laboratories), Streptavidin Sepharose High Performance (GE Healthcare), Streptavidin Polystyrene Microspheres (Microspheres Nanospheres), Streptavidin Coated Polystyrene Particles (Spherotech), or any other streptavidin coated bead or resin commonly used by one skilled in the art to capture biotin-tagged molecules.
In this specification the term "partition" refers to separation or removal of one or more molecular species, complexes or conjugates. Partitioning of free peptide from peptide- cargo construct conjugates is effective following formation of peptide-cargo construct conjugates. The partitioning step may separate peptide-cargo construct conjugates bound to a capture element, thereby forming a complex of cargo construct and capture element, from peptide not bound to capture element. Partitioning may involve immobilization of peptide-cargo conjugates on a solid support thereby separating and purifying the conjugates. Partitioning may also involve separation of peptide-cargo conjugates from unwanted contaminants.
The methods of forming peptide-cargo conjugates or generating peptide libraries according to this disclosure may comprise one or more optional wash steps, e.g. after formation of peptide-cargo constructs, or following partition of reagents/products.
In this specification the release of peptide-cargo conjugate from the tag refers to the separation of the conjugated cargo molecule and peptide from the tag and any capture element with which it is associated. Release may involve cleavage of the tag and/or cleavabie linker to separate the peptide-cargo molecule construct from the tag and/or cleavabie linker. Release may therefore involve a step of cleavage of the peptide-cargo conjugate from the tag and/or linker. The release may provide free peptide-cargo conjugates, which may be collected, isolated and/or purified to provide a composition comprising isolated and/or substantially purified peptide-cargo conjugates. Some aspects and embodiments of the present invention refer to a plurality of peptide- cargo conjugates, or a plurality of peptide-cargo construct conjugates. In this specification a plurality refers to at least two, but preferably refers to one of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 1 1 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more.
In some aspects and embodiments of the present invention synthesis reactions are performed in parallel. This refers to performing a plurality of synthesis reactions simultaneously thereby allowing for the generation of a plurality of different peptide-cargo conjugates, or peptide-cargo construct conjugates in about the time required to complete a single synthesis reaction.
Parallel reactions may be performed side-by-side, e.g. in a plurality of adjacent reaction chambers. In one embodiment a multi-well plate, e.g. 12, 48 or 96 well plate may be used in which individual reactions are performed in separate wells of the plate. Synthesis reactions or screening may be automated, e.g. the method, experiment or reaction may be performed or conducted without manual human intervention being performed by a machine or robot under the control of a suitably programmed computer.
In some aspects and embodiments of the present invention reactions or assays are performed simultaneously. This refers to performing the reactions or assays at the same time, rather than consecutively, such that a plurality of reactions or assays may be completed in about the time required to complete a single reaction or assay. This permits the high-throughput synthesis of conjugates and high-throughput screening of conjugates required during the process of drug discovery or validation.
In some aspects and embodiments of the present invention assays of peptide-cargo conjugates are performed in respect of a functional property. The functional property may be any such property of interest. In particular the assay may test the ability of the peptide to act as a cell penetrating peptide (CPP) [a CPP screening assay] in combination with its cargo. Such assays are well known (e.g. the HeLa pLuc705 cell assay described herein) and generally test for the ability of the peptide to facilitate uptake of the cargo molecule into a cell. The uptake may be assessed against a control comprising the cargo molecule not conjugated to the peptide.
Some aspects and embodiments of the present invention concern a library of peptide- cargo conjugates. In this context, a library refers to a collection of different peptide-cargo conjugates, each stored individually, e.g. in an individual container, but also as part of a collection in which the plurality of peptide-cargo conjugates/containers are stored in one geographical location, e.g. one or more cold stores (fridges, freezers) in the same building. Each container may contain a sufficient quantity of isolated or purified peptide- cargo conjugate to permit a plurality of samples to be taken, e.g. to allow use in multiple function assays or screening methods. As such, each container may contain a quantity of peptide-cargo conjugate in the range of 10-1000ng or 1-1000Mg or 1-1 Omg.
The library may contain a plurality of series' of conjugates. Each series comprises a plurality of peptide-cargo conjugates, as described above. Within a given series the cargo molecule is the same for each conjugate but the peptide is different between each conjugate. Between series the cargo molecule is different and the conjugates within a series differ in respect of the peptide component and preferably have a range of peptide components that corresponds to the other series in the set. A library may have one or a plurality of different sets of series of conjugates, e.g. the sets differing in terms of the composition of the peptide components. In aspects and embodiments of the present invention compositions comprising isolated and/or purified or substantially purified peptide-cargo construct conjugates or peptide- cargo conjugates are provided. Such compositions are enriched for the respective conjugate, and may be free or substantially free of unreacted peptide (e.g. less than one of 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % of the peptide component of the composition is free or unreacted compared with the conjugated peptide component) and/or of unreacted cargo.
Methods according to the present invention are preferably performed in vitro. The term "in vitro" is intended to encompass experiments with reagents, reactants, materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term "in vivo" is intended to encompass experiments and procedures with intact multicellular organisms. Where the method is performed in vitro it may comprise a high throughput screening assay. Test compounds used may be obtained from a synthetic combinatorial peptide library, or may be synthetic peptides or peptide mimetic molecules. Other test compounds may comprise defined chemical entities, oligonucleotides or nucleic acid ligands. In another embodiment a screening assay may be performed in vivo where only a small quantity (e.g. μgs) of conjugate is required to be delivered (e.g. by intramuscular delivery) into an animal such as a mouse. In some preferred embodiments the peptide is a cell penetrating peptide (CPP;
sometimes called membrane translocating peptides or cell delivery peptides), or a candidate cell penetrating peptide - that is, a peptide designed/synthesised for assessment of its activity as a cell penetrating peptide. Cell penetrating peptides are peptides that are useful in facilitating the uptake of a conjugated cargo molecule, e.g. where the cargo molecule is useful as a therapeutic but requires uptake to a cell in order to exert its therapeutic effect.
For example, such peptides are useful in the case of oligonucleotide analogues useful as steric blocking agents such as those with uncharged backbones, such as peptide nucleic acids (PNA) [Egholm M., Buchardt O., Nielsen P. E., Berg R. H.: J. Amer. Chem. Soc. 1992, 114, 1895] and phosphorodiamidate morpholino oligonucleotides (PMO) [Summerton J., Weller D.: Antisense Nucl. Acid Drug Dev. 1997, 7, 187]. Both PNA and PMO ONs have been used in vivo for RNA targeting applications towards the
development of therapeutics [Lebleu B., Moulton H. M., Abes R., Ivanova G. D., Abes S., Stein D. A., Iversen P. L, Arzumanov A., Gait M. J.: Adv. Drug Delivery Rev. 2008, 60, 517]. In cell culture, both PNA and PMO are observed to enter cells only rather poorly and therefore much effort has been expended to develop methods of enhancing cell delivery.
A range of CPPs are known. Although some known CPPs share common sequence structure characteristics, a single set of such characteristics does not define a CPP per se. A candidate CPP may have any amino acid sequence. Indeed, the present invention is useful in investigating sequence structure of new candidate CPPs as well as in investigating sequence variation of known CPPs. Known CPPs are described in WO201 1/064552 WO2009144481 , WO2009/147368 and WO2013/030569, each of which are incorporated by reference in their entirety.
By way of example, known CPPs include Penetratin, Tat, Transportan, and (R-Ahx-R)4
(Ahx = aminohexanoic acid), and R6-Penetratin (R6Pen) [Zatsepin T. S., Turner J. J., Oretskaya T. S., Gait M. J.: Curr. Pharm. Design 2005, 1 1 , 3639; Abes R., Arzumanov A.,
Moulton H. M., Abes S., Ivanova G. D., Iversen P. L, Gait M. J., Lebleu B.: Biochem.
Soc. Trans. 2007, 35, 775; Turner J. J., Arzumanov A., Ivanova G., Fabani M., Gait M. J.:
Cell-Penetrating Peptides, 2nd Edition. (U. Langel Ed.) 2006, CRC Press, Boca Raton.
313; Turner J. J., Jones S., Fabani M., Ivanova G., Arzumanov A., Gait M. J.: Blood, Cells, Molecules and Diseases 2007, 38, 1 ; Turner J. J., Ivanova G. D., Verbeure B.,
Williams D., Arzumanov A., Abes S., Lebleu B., Gait M. J.: Nucl. Acids Res. 2005, 33,
6837].
Abes et al (Nucleic Acids Research, 2008, 36, 6353-6354) discuss CPP molecules having an (RXR)n-PMO structure and reports that of several spacer (X) molecules tested, a linear C4 (Abu), C6 (Ahx), or Cs (Acy) spacer is most effective.
Saleh et al (Bioconjugate Chem. Vol. 21 , No.10 1902- 91 1 (2010)) reported that increasing the number of Arginine residues in a linear (RXR)n arrangement to 12 and 16 for conjugates with PNA increased the splicing correction ability in a HeLa cell assay, but also increased cell toxicity. In some cases a class of CPP can be defined as a peptide having a primary sequence structure comprised of at least three domains, having the arrangement:
N-terminus [Domain 1 ] - [Domain 2] - [Domain 3] C-terminus
wherein the number of R (Arginine) residues in Domains 1 and 3 combined is at least 5, the number of X residues in Domains 1 and 3 combined is at least 1 , the number of B residues in Domains 1 and 3 combined is at least 2, wherein X= one of 6-aminohexanoic acid, 4-aminobutyric acid, aminocaprylic acid, p-aminobenzoic acid or isonipecotic acid, and B=betaAlanine, and wherein Domain 2 comprises a sequence that contains at least 3 of the amino acids Z1Z2FLI, where Zi is Y or I and Z2 is Q or R.
In another example a class of CPP can be defined as a peptide having a primary sequence structure comprised of at least three domains, having the arrangement::
N-terminus [Domain 1 ] - [Domain 2] - [Domain 3] C-terminus
in which Domain 1 comprises a sequence chosen from RXRRBRRXR, RBRRXRRBR, RXRRXR, Domain 2 comprises a sequence chosen from: ILFQ, ILIQ, Domain 3 comprises a sequence chosen from RXRBRXR, RBRXRBR, RXRRXR, RXRXRXR, RXRBRX, MKWHK, WKWHK, in which X= one of 6-aminohexanoic acid, 4-aminobutyric acid, aminocaprylic acid, p-aminobenzoic acid or isonipecotic acid, and B=betaAlanine.
In yet another example a class of CPP can be defined as a peptide having a primary sequence structure comprised of at least three domains, having the arrangement:
N-terminus [Domain 1] - [Domain 2] - [Domain 3] C-terminus
in which Domain 1 comprises a sequence chosen from RXRZ3 where Z3 is selected from one of: RBRRXR, RBRRX, RBRX, RBRXR, BRX, BX, RBR, RB, or RBRR, Domain 2 comprises a sequence that contains at least 3 of the amino acids Z-|Z2FLI, where Zi is Y or I and Z2 is Q or R, Domain 3 comprises a sequence chosen from RXRBRXRB, XRBRXRB, RXRRBRB, BRXRB, XRRBRB, RRBRB, XRBRB , RBRXRB, RXRBRB, or BRBRB in which X= one of 6-aminohexanoic acid, 4-aminobutyric acid, aminocaprylic acid, p-aminobenzoic acid or isonipecotic acid and B=betaAlanine.
Yet further examples of known CPPs include MSP (ASSLNIA), AAV6
(TVAVNLQSSSTDPATGDVHVM), the AAV8 IVADNLQQQNTAPQIGTVNSQ, TAT (YGRKKRRQRRRP) or (RXR)4 (RXRRXRRXRRXR) [where R is L-arginine and X is 6- aminohexanoic acid], and suitable peptides may comprise the sequences.
In yet other embodiments a CPP may comprise a sequence of the formula (RZR(Z)i(ILFQY)m)n or a functional derivative thereof, wherein Z is an aminoalkyi spacer, I is 0 or 1 , m is 0 or 1 and n is from 2 to 6, and I = isoleucine, L = leucine, F =
phenylalanine, Q = glutamine, Y = tyrosine. An aminoalkyi spacer is typically a molecule that can separate amino acids in the peptide chain. The aminoalkyi spacer may have from 1 to 6, such as 2, 3, 4 or 5, carbon atoms. The aminoalkyi spacer typically comprises an amino group and a carboxyl group such that it can bind to the adjacent amino acids in the peptide chain through peptide bonds. Preferred aminoalkyi spacers include, but are not limited to, 6-aminohexanoyl (X), betaalanyl (B), 4-aminobutyryl, p-amino benzoyl, or isonipecotyl.
A candidate CPP may comprise two or more RZR groups (for example RXR and/or RBR groups). The number of these groups being determined by the value of n, where n is from 2 to 6, such as 3, 4 or 5. For each value of n, the Z in RZR may independently be X or B. For instance, the peptide may comprise the sequence RXRRXR, RBRRBR, RXRRBR or RBRRXR. The two or more RXR and/or RBR groups may be separated by Z (if I is 1 ) and/or ILFQY (if m is 1 ). For each value of n, if I is 1 , m is preferably 0. For each value of n, if m is 1 , I is preferably 0. For each value of n in a positively charged peptide, the separating group may independently be Z or ILFQY. For instance, if n is 3, the peptide may comprise the sequence RXRZRXRILFQYRXR (i.e. where the first two RZRs are separated by Z and the second two RZRs are separated by ILFQY).
Yet further examples of CPPs and candidate CPP sequences are set out in Figures 10 to 14. Accordingly, in some embodiments the invention concerns the provision and/or formation and/or screening of a peptide-cargo construct comprising a cell delivery peptide (the peptide), or candidate cell delivery peptide, covalently or non-covalently attached to a biologically active compound (the cargo). Peptide-cargo conjugates according to the present invention may be provided for use in a method of medical treatment. The medical treatment may preferably require delivery of the cargo molecule into a cell and optionally the nucleus of the cell.
Peptides and/or peptide-cargo conjugates are accordingly provided for use in treatment of disease. The use of a peptide and/or a peptide-cargo conjugate in the manufacture of a medicament for the treatment of disease is also provided. A method of treatment of a patient or subject in need of treatment for a disease condition is also provided comprising the step of administering a therapeutically effective amount of a peptide and/or a peptide- cargo conjugate to the patient or subject. Preferably, the cargo component of a peptide- cargo conjugate comprises an active agent (e.g. pharmaceutical agent) capable of treating, preventing or ameliorating the disease.
Diseases to be treated may include any disease where improved penetration of the cell and/or nuclear membrane by a pharmaceutical or therapeutic molecule may lead to an improved therapeutic effect. Diseases to be treated may include disease conditions caused by (in whole or in part) splicing deficiencies, e.g. Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy, and other muscle diseases such as limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculpharyngeal muscular dystrophy (OMD), distal muscular dystrophy and Emery-Dreifuss muscular dystrophy (EDMD), as well as Menkes
Disease38, β-thalassemia39, splice correction of tau protein to relieve frontotemporal dementia, parkinsonism and spinal muscular atrophy39, Hutchinson-Gilford Progeria Syndrome40, Ataxia-telangiectasia mutated (ATM)41, spinal muscular atrophy, myotonic dystrophy 1 , or cancer. Genes implicated in the pathogenesis of some of these diseases include dystrophin (Duchenne muscular dystrophy and Becker muscular dystrophy), DMPK (DM1 type MD), ZNF9 (DM2 type MD), PABPN1 (OMD), emerin, lamin A or lamin C (EDMD), myotilin (LGMD-1A), lamin A/C (LGMD-1 B), caveolin-3 (LGMD-1 C), calpain-3 (LGMD-2A), dysferlin (LGMD-2B and Miyoshi myopathy), gamma-sarcoglycan LGMD- 2C), alpha-sarcoglycan (LGMD-2D), beta-sarcoglycan (LGMD-2E), delta-sarcoglycan (LGMD-2F and CMD1 L), telethonin (LGMD-2G), TRIM32 (LGMD-2H), fukutin-related protein (LGMD-2I), titin (LGMD-2J), and O-mannosyltransferase-1 (LGMD-2K).
In such cases of diseases involving splicing defects the cargo may comprise an oligonucleotide, PNA, PMO or other oligonucleotide types, including LNA, capable of preventing or correcting the splicing defect and/or increasing the production of (e.g. number of) correctly spliced mRNA molecules. The present invention is, of course, not limited to cargo molecules capable of correcting a splicing defect. Cargo molecules may include other oligonucleotide, PNA, PMO or LNA molecules such as oligonucleotide molecules capable of targeting mRNA or microRNA, e.g. siRNA or shRNA for knockdown of gene expression, as well as non-oligonucleotide molecules (such as peptides). The patient or subject to be treated may be any animal or human. The patient or subject may be a non-human mammal, but is more preferably a human patient. The patient or subject may be male or female. Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural,
intraperitoneal, subcutaneous, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for
administration by injection to a selected region of the human or animal body.
In accordance with the present invention methods are also provided for the production of pharmaceutically useful compositions, which may be based on a peptide-cargo conjugate formed, screened, assayed or tested as described herein. In addition to the steps of the methods described herein, such methods of production may further comprise one or more steps selected from:
(a) identifying and/or characterising the structure of a selected peptide-cargo conjugate;
(b) obtaining or providing the peptide-cargo conjugate;
(c) mixing the selected peptide-cargo conjugate with a pharmaceutically
acceptable carrier, adjuvant or diluent.
For example, a further aspect of the present invention relates to a method of formulating or producing a pharmaceutical composition, the method comprising identifying a peptide- cargo conjugate in accordance with one or more of the methods described herein, and further comprising one or more of the steps of:
(i) identifying the peptide-cargo conjugate; and/or
(ii) formulating a pharmaceutical composition by mixing the selected peptide- cargo conjugate or a prodrug thereof, with a pharmaceutically acceptable carrier, adjuvant or diluent.
Certain pharmaceutical compositions formulated by such methods may comprise a prodrug of the selected substance wherein the prodrug is convertible in the human or animal body to the desired active agent. In other cases the active agent may be present in the pharmaceutical composition so produced and may be present in the form of a physiologically acceptable salt. The designing of mimetics to a known pharmaceutically or biologically active compound is a known approach to the development of pharmaceuticals and therapeutics based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. some peptides may be unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".
Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic. A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
With regard to the present invention, a method is provided comprising the step of modifying the peptide structure and conjugating to a peptide or oligonucleotide cargo, such as PNA or PMO, optionally followed by testing the modified peptide-cargo conjugate in a splice correction assay or an exon skipping assay and/or in a cell viability assay. This process of modification of the peptide or peptide mimetic conjugate may be repeated a number of times, as desired, until a peptide-cargo conjugate having the desired splice correction or exon skipping activity and/or cell viability is identified.
The modification steps employed may comprise truncating the peptide or peptide mimetic length (this may involve synthesising a peptide or peptide mimetic of shorter length), substitution of one or more amino acid residues or chemical groups, and/or chemically modifying the peptide or peptide mimetic to increase cell viability of its corresponding cargo conjugate, resistance to degradation, transport across cell membranes and/or resistance to clearance from the body and/or to provide activity in exon skipping and dystrophin production in an mdx mouse model of DMD, including in heart muscle. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Brief Description of the Figures
Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which: Figure 1. Diagrammatic overview of conjugation and conjugate work-up strategy of SELPEPCON.
Figure 2. Chart showing fold increase in luminescence caused by PNA705 conjugates of LB 1 -peptides compared to a buffer blank induced by the conversion of Beetle luciferin to oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 μΜ conjugate concentration.
Figure 3. Chart showing fold increase in luminescence caused by PNA705 conjugates of LB2-peptides compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 μΜ conjugate concentration.
Figure 4. Chart showing fold increase in luminescence caused by PNA705 conjugates of LB2-peptides obtained by SELPEPCON (Crude) and RP-HPLC purified (Purified) compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 μΜ conjugate concentration. Figure 5. Chart showing fold increase in luminescence as a function of a) the net charge and b) the number of arginines of the CPPs in the LB2-PNA705 conjugates compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 5 μΜ conjugate concentration. The dots represent the data points and the line represents the average.
Figure 6. RT-PCR analysis to measure aberrant and redirected RNA levels in HeLa pLuc705 cells after exposure to CPP-PNA705 conjugates at different concentrations. The activity is presented as a percentage of corrected RT-PCR product obtained from splicing redirection.
Figure 7. Scheme 1 : Synthesis of N3-PNA705-S-S-biotin cargo for conjugation to CPP-libraries
Figure 8. Scheme 2: Synthesis of alkyne functionalized peptides for conjugation to N3-PNA705-S-S-biotin Figure 9. Scheme 3: Outline of conjugation and purification strategy to obtain CPP- PNA705 conjugate libraries Figure 10. Table 1 : Library of Pip5h analogues containing an A/-terminal alkyne linker, peptide and conjugate synthesis yield.
Figure 11. Table 2: MALDI-TOF mass-spectrometry analysis of LB1 -peptides and their PNA705 conjugates synthesized using SELPEPCON
Figure 12. Table 3: Sequences and MALDI-TOF mass-spectrometry analysis of LB2 CPP-library containing a A erminal alkyne linker and conjugation to N3-PNA705-S-S- biotin. Figure 13. Table 4: Sequences and MALDI-TOF mass-spectrometry analysis of LB2 CPP-library containing a /V-terminal alkyne linker and conjugation to N3-PNA705-S-S- biotin.
Figure 14. Table 5: Sequences and MALDI-TOF mass-spectrometry analysis of LB2 CPP-library containing an A/-terminal alkyne linker and conjugation to N3-PNA705-S-S- biotin.
Figure 15. Table 6: : Yields of parallel peptide and PNA705-conjugate synthesis of LB2-CPP-Library.
Figure 16. Table 7: Yields of parallel peptide and PNA705-conjugate synthesis of LB2-CPP-Library.
Figure 17. Table 8: Yields of parallel peptide and PNA705-conjugate synthesis of LB2-CPP-Library.
Figure 18. MALDI mass-spectra of parallel-synthesized LB1 - and LB2-peptides obtained by SELPEPCON. In (A) to (L) examples are given of MALDI-TOF mass-spectra of LB1 - and LB2-peptides obtained using the SELPEPCON parallel synthesis procedure. (K) and (L) show peptides that showed several peaks belonging to impurities. (A) MALDI- TOF mass-spectrum of LB1_1 . (B) MALDI-TOF mass-spectrum of LB1_12. (C) MALDI- TOF mass-spectrum of LB2_3. (D) MALDI-TOF mass-spectrum of LB2_5. (E) MALDI- TOF mass-spectrum of LB2_7. (F) MALDI-TOF mass-spectrum of LB2_24. (G) MALDI- TOF mass-spectrum of LB2_27. (H) MALDI-TOF mass-spectrum of LB2_54. (I) MALDI- TOF mass-spectrum of LB2_68. (J) MALDI-TOF mass-spectrum of LB2_72. (K) DI-TOF mass-spectrum of LB2_40. (L) MALDI-TOF mass-spectrum of LB2_5 .
Figure 19. HPLC graphs of parallel-synthesized peptides obtained by SELPEPCON. These were recorded on a Phenomenex analytical C18 Jupiter column (250 x 4.6 mm, 5 micron) using the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 15% B 2-20 min 15%-30% B 20-25 min 30%-90% B. (A) HPLC-graph of the LB1_5 obtained by SELPEPCON. (B) HPLC-graph of the LB1_10 obtained by SELPEPCON.
Figure 20. MALDI mass-spectra of LB1 - and LB2-PNA705 conjugates obtained by SELPEPCON. In (A) to (M), examples are given of MALDI-TOF mass-spectra of conjugates obtained using the SELPEPCON procedure. (A) to (L) show MALDI-TOF mass-spectra of PNA705 conjugates from libraries LB1 and LB2 that were tested in the splicing redirection assay. As an example Figure (M) shows a MALDI-TOF mass- spectrum of a conjugate for which the conversion of the conjugation reaction was low and was thus not tested. Figure (K) shows an example of a conjugate containing a cysteine in the CPP-sequence for which the mixture of conjugate containing different amounts of StBu groups was found by MALDI-spectroscopy. (A) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB1_4. (B) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB1_8. (C) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB1_1 1. (D) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_27. (E) MALDI-TOF mass- spectrum of the PNA705 conjugate of LB2_28. (F) MALDI-TOF mass-spectrum of the
PNA705 conjugate of LB2_35. (G) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_38. (H) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_43. (I) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_44. (J) MALDI-TOF mass- spectrum of the PNA705 conjugate of LB2_54. (K) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_68. (L) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_71. (M) MALDI-TOF mass-spectrum of the PNA705 conjugate of LB2_31.
Figure 21. HPLC graphs of CPP-PNA705 conjugates obtained by SELPEPCON. (A) and (B) are example HPLC graphs of LB2-PNA705 conjugates. These were recorded on a Phenomenex analytical C18 Jupiter column (250 x 4.6 mm, 5 micron) using the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 10% B 2-20 min 10%-30% B 20-30 min 30%-50% B 30-35 min 50%-90% B. Unconjugated PNA impurities (±10%) can be observed which are likely the results of some impurities in the PNA that do not contain the azide functional group. In addition, impurities are observed that share a mass close to that expected of the conjugate. These can be the result of either impurities in the peptide or degradation of the conjugate. (A) HPLC-graph of the LB2_21-PNA705 conjugate obtained by SELPEPCON. (B) HPLC-graph of the LB2_22-PNA705 conjugate obtained by SELPEPCON.
Figure 22. Chart showing fold increase in luminescence caused by PNA705 conjugates of LB1 -peptides obtained by SELPEPCON (Crude) and RP-HPLC purified (Purified) compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells at 1 and 2 μΜ conjugate concentration. Figure 23. Chart showing fold increase in luminescence caused by PNA705 conjugates of LB2-peptides compared to a buffer blank induced by the conversion of Beetle luciferin into oxyluciferin by expressed luciferase via splicing redirection in HeLa pLuc705 cells in a single point screening assay at 5 μΜ conjugate concentration. Figure 24. Table 9. Library of LB1 peptides containing a C-terminal alkyne, peptide and conjugate synthesis yields.
Figure 25. Table 10 MALDI-TOF mass-spectrometry analysis of C-terminal alkyne containing LB -peptides and their PNA705 conjugates synthesized using SELPEPCON.
Figure 26. Table 1 1. Synthesized PMI and 17 28P53 peptides and MDM2 binding capacity in vitro.
Figure 27. Table 12. Conjugation of LB2 CPP-peptides containing an /V-terminal alkyne linker to azide modified PMI constructs.
Figure 28. Diagram illustrating synthesis of bifunctional PMO-cargo.
Figure 29. Diagram illustrating the conjugation of biotinylated peptides containing a CXB C-terminal extension to PMO via SELPEPCON and the subsequent capping of conjugates using iodoacetamide. To demonstrate the utility of this SELPEPCON methodology, two case studies involving a PNA cargo are shown. In the first example, a small conjugate library is synthesized to investigate the roles of individual or groups of amino acids in a pre-selected CPP attached to a cargo. The second shows a larger CPP screen of a variety of quite different peptide sequences to find CPP candidates suitable for delivery of a novel cargo into a cell. Both studies utilize CPP conjugates of a PNA705 cargo, a well-known 16-mer splice-redirecting oligonucleotide analogue cargo that has been utilized many times for CPP development in the past.6 A HeLa pl_uc705 cell assay is available for this cargo, which utilizes splicing redirection of an aberrant β-globin intron and which leads to the up- regulation of firefly luciferase.7 The assay is convenient, has a high dynamic range and is very suitable for testing of a large number of conjugates in parallel.
Conjugations are carried out using copper catalyzed Huisgen "click" reactions8 between alkyne-functionalized CPPs and an azide-functionalized PNA705 (sequence:
CCTCTTACCTCAGTTACA)containing a disulphide-linked biotin-tag. A disulphide-linked biotin tag allows for solid-phase immobilization purification of the resultant conjugates after which the conjugates can be isolated by reduction of the disulphide, which releases it from the solid support. The results demonstrate how SELPEPCON can be utilized to find active CPPs for a cargo such as PNA705 in a rapid synthesis, isolation and screening procedure.
Examples Example 1
Experimental General
MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro
BioSpectrometry workstation. A stock solution of 10 mg mL'1 of
a-cyano-4-hydroxycinnamic acid in 60% acetonitrile in water was used as matrix. Error bars are ± 0.1 %. Reversed phase HPLC purifications and analyses were carried out using a Varian 940-LC. A Nanodrop 2000 UV analyser (Thermo Scientific) was used for the quantification of PNA concentrations using the absorption at 260 nm with the following values to calculate absorption coefficients; C 6.6 cm - M~1, T 8.6 crrr M-1, A 13.7 cm_1M"1, G 1 1.7 crrr1M~1. TBTA (frs-(benzyltriazolylmethyl)amine) was synthesised according to a literature procedure.14 The synthesis of the Pip-1 -PNA705 conjugate had been reported previously.6' 13b N3-PNA705-SH synthesis
The PNA705 sequence containing a C-terminal Cys and two flanking Lys residues (Lys- CCTCTTACCTCAGTTACA-Lys-Cys) was synthesised on a 50 μηποΙ scale using a modified Liberty Peptide Synthesizer (CEM) according to a published procedure [10] using a Chem-Matrix solid support and Fmoc amino acid monomers (Novabiochem) or Fmoc (Bhoc) PNA monomers (Link Technologies). After the final deprotection, the support was removed from the Synthesizer and 5-azidopentanoic acid was manually double coupled using a standard PyBop/NMM coupling reaction at room temperature for 2 x 15 min. The PNA was cleaved from the support and deprotected using TFA (1.5 mL) containing 10% triisopropylsilane/2.5% water and 1 % phenol. The mixture was agitated for 2 h after which it was filtered and concentrated. The crude PNA was isolated by cold diethyl ether precipitation, dissolved in water, filtered and purified by HPLC. A
Phenomenex Prep C18 Jupiter column (250 x 21.2 mm, 10 micron) was used with the following gradient (A: 0.1 % TFA, B: 90% acetonitnle, 0.1 % TFA) 0-2 min 7.5% B 2-20 min 7.5%-25% B 20-25 min 25%-90% B (retention time: 20.4 min). The fractions containing the desired PNA were combined and freeze-dried giving a fluffy white solid (yield 10-40% based on support loading). Mass, expected: m/z 5250.0 found: m/z 5256.9.
N3-PNA705-S-S-biotin construct
To a solution of 4 pmol PNA705 in 3 ml water, was added 8 μιηο! EZ-link HPDP-Biotin (Thermo Scientific) from a 3.7 μηηοΙ ml"1 stock in DMSO. To this solution 600 μΙ 2M sodium acetate pH 7 was added and the resulting solution shaken for two hours. The reaction was quenched by the addition of 15 ml 0.1 % TFA and 150 μΙ TFA in order to solubilise the precipitate formed. This solution was filtered and then purified by HPLC using a Phenomenex Prep C 8 Jupiter reversed phase column (250 x 21.20 mm, 10 micron) with the following gradient (A: 0.1 % TFA, B: 90% MeCN 0.1 % TFA) 0-2 min 12% B 2-25 min 12%-25% B 25-30 min 25%-90% (Retention time: 25.0 min). The purified N3- PNA-705-S-S-biotin was obtained as a fluffy white powder in a typical yield of 60-80%. Mass, expected: m/z 5678.4 found: m/z 5687.1.
Peptide synthesis Peptide library synthesis was carried out on a 5 prnol scale using an Intavis Parallel Peptide Synthesizer, applying standard Fmoc chemistry and following manufacturer's recommendations. The solid support was as supplied by Intavis (Tentagei, 0.2 mmol g ). Double coupling steps were used with a PyBop/NMM coupling mixture followed by acetic anhydride capping after each coupling step. Terminal alkyne functionalization involved a standard coupling procedure using 4-pentynoic acid in the final step of the synthesis. The peptides were cleaved from the support and deprotected by addition of TFA (1.5 mL) containing 5% triisopropylsilane/2.5% water and 1 % phenol with shaking for 4 h. The support suspension was then concentrated to a volume of ±500 μΙ_ using a flow of nitrogen and diluted with 5 mL water. After mixing, the resulting mixture was loaded on a 20cc Oasis HLB cartridge (Waters), which was previously washed with acetonitrile (10 mL) and equilibrated with 0.1 % TFA (3 x 10 mL). After loading, the cartridge was washed with 0.1 % TFA (2 x 10 mL) and 5% acetonitrile in 0.1 % TFA (2 x 0 mL). The peptide was eluted with 40% acetonitrile (10 mL). The solution obtained was freeze-dried and the yield was calculated using the weight obtained (Table 1 , Figure 10), corrected for the amount of TFA salts based on the number of positive charges present in the peptide. For mass spectroscopy data see Table 2, Figure 1 1.
Conjugate synthesis
A mixture was prepared containing 30 nmol PNA from a stock solution in water (±2 mM), 150 nmol peptide from a stock solution in NMP (± 10 mM), 0.2 μΙ 2,6-lutidine (1.7 μητιοΙ) and 1 μΙ diisopropylethylamine (5.7 μιτιοΙ). 7.5 μΙ of 20 mM CuSCVTBTA solution (150 nmol) premixed in a 1 :1 mixture of DMSO and 10 μΙ of a 20 mM solution of sodium ascorbate (200 nmol) were added to this mixture. The mixture was left for one hour and quenched with 100 μΙ 0.2 M EDTA 40% acetonitrile and 800 μΙ TBS 40% acetonitrile. The solution obtained was vortexed and loaded in two batches of 500 μΙ on a streptavidin HP SpinTrap (GE Healthcare UK Limited) and each batch was incubated for 20 minutes while being mixed by inversion. The column was washed with 400 μΙ 0.1 M EDTA in TBS 40% acetonitrile and with 5x 400 μΙ TBS 40% MeCN. The conjugate was released from the resin by reaction for with 2x 20 minutes with 2x 400 μΙ 10 mM TCEP in TBS 40% acetonitrile and the solid support washed with 200 μΙ 40% acetonitrile in TBS. The solutions collected were combined and freeze-dried. The solid was dissolved in 500 μΙ 20% acetonitrile and loaded on an equilibrated 1 cc Oasis HLB cartridge (Waters) together with 500 μΙ 0.1 % TFA. The column was washed with 3x 1 ml 0.1 % TFA, 3x 1 ml 5% acetonitrile 0.1 % TFA and 1x 1 ml 10% acetonitrile 0.1 % TFA. These extensive washes are required to remove the TCEP from the cartridge. The conjugates are released using 500 μΙ 60% acetonitrile and diluted with 50 μΙ 0.1 % TFA. The resulting solution was freeze-dried and dissolved in 500 μΙ water. The concentration was determined and the solution was stored in the freezer for use in cellular assays. Splice-redirection assay
The splicing redirection assay was carried out similarly to a previously reported procedure with minor changes.6 The assay was performed using 48-well plates with cells seeded the previous day (7.5x104 cells per well) and a total volume 100 μΙ per well for the conjugates' solutions in OptiMEM. RT PGR experiments were carried out as reported in previous publications.120 All experiments were performed in triplicates, unless specifically stated as designed one-point experiments.
Results and Discussion Cargo synthesis
The SELPEPCON method of synthesis of a bifunctionalized PNA705 cargo suitable for attachment of peptides and subsequent immobilization is shown in Scheme 1 , Figure 7. Since a known side product of the click reaction is alkyne homocoupling, it is better to locate the azide functionality on the PNA cargo and the alkyne functionality on the peptide component, since any alkyne-homocoupling side reaction will lead to a product that is easily removed by washing of the streptavidin solid support. Thus, 5-azidopentanoic acid was used for N-terminal modification of the PNA by manual solid phase amide coupling following machine-aided assembly of the PNA by solid-phase synthesis.9 The PNA contains a C-terminal S-trityl-protected Cys residue, which was introduced at the first step of solid-phase synthesis. After deprotection and cleavage from the solid support, a biotin group was introduced on to the N3-PNA705 by disulfide bridge formation via reaction of the C-terminal Cys residue with commercially available Λ/-[6- (biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (EZ-link™ HPDP Biotin). This straight forward reaction provided the N3-PNA(705)-S-S-biotin after HPLC purification in 80% yield.
Pip5h based CPP-library (LB1)
In order to demonstrate the methodology, a small test library of 16 peptides was synthesized on solid phase using an INT AVIS Parallel Peptide Synthesizer (Table 1 ,
Figure 10). The test library was narrowly based around a known relatively short (17 amino acids) CPP, Pip5h, which we have previously evaluated in exon skipping as a conjugate with a PMO targeting the pre-mRNA of dystrophin10, but which was not chosen for further development. Peptide variants included modifications in the central 5-amino acid core (LB1_1 - 8) or on one of the flanking Arg-rich flanking regions (LB1_9 - 16) of the sequences. In each case an /V-terminal alkyne was introduced using 4-pentynoic acid through standard amide bond formation as the final coupling step (Scheme 2, Figure 8).
The peptides were deprotected and cleaved from the solid support by standard TFA treatment for 4 h with gentle mixing and rapidly isolated by reversed phase OASIS HLB cartridge purification. Good yields were achieved using this synthesis and purification method (Table 1 , Figure 10). MALDI-TOF mass-spectrometry of the products showed in all cases a major signal corresponding to the expected peptide and only minor amounts of peptide impurities (Table 2, Figure 11 and Figures 18(A), 18(B) and 19(A), 19(B)). None of the observed shorter peptide impurities should form conjugates in the
subsequent conjugation step because the alkyne linker is introduced in the final step of the synthesis and truncated peptides are capped after each coupling step during solid phase synthesis. Thus, time-consuming HPLC purification of each synthesized peptide is avoided. The peptide purification method used is fast, such that cleavage, deprotection and purification of 16 peptides can be carried out within one day, thus allowing for easy parallelization.
LB1-PNA705 conjugate library synthesis
Peptide-PNA conjugates were synthesized, purified and isolated in parallel using the procedure shown in Scheme 3, Figure 9. Copper(ll) sulphate in combination with ascorbic acid proved to be the most promising catalyst system. Procedures involving copper(l) salts, such as copper(l) bromide or copper(l) iodide, were also possible but provided less reproducible conversions. Di-isopropylethylamine was used as base to quench TFA salts of the cargo and peptides, and 2,6-lutidine and £r/s(benzyltriazolylmethyl)amine (TBTA) were utilized to stabilize the active copper(l) species. After 1 h, reactions were quenched by the addition of EDTA and diluted, after which the conjugates were immobilized on a streptavidin-functionalized solid support held in SpinTrap™ centrifuge tubes. After washing, the conjugates were released from the support by addition of a TBS buffer solution containing 0 mM £r/s(2-carboxyethyl)phosphine (TCEP) followed by
centrifugation, leaving the biotin tag on the support. The use of organic solvent (e.g. 40% acetontrile) for dilution of the reaction mixtures, washing and release proved to be essential for good recovery of the conjugates. The resultant conjugates still contain buffer and TCEP, which were removed by reversed phase OASIS HLB cartridge purification. The conjugates were obtained in good isolated yields (50-70%, Table 1 , Figure 10). In all cases, analysis by MALDI-TOF
mass-spectrometry showed the expected signal for the conjugation product (Table 2, Figure 11 and Figures 20(A) - 20(B)). In addition, a signal corresponding to the unconjugated PNA705 was observed. Quantitative analysis by RP-HPLC revealed <10% of the unconjugated cargo. Because the cargo by itself is unable to enter cells efficiently and has been demonstrated to be inactive in the HeLa pLuc705 cell splicing redirection assay (data not shown), the mixtures were used without further purification.
Compared to standard methods of preparing CPP-PNA conjugates, SELPEPCON is very fast and convenient. For a library of 16 conjugates, parallel peptide synthesis and purification took 3 days, and conjugation and purification took only two days including lyophilisation time. Thus conjugates were available within a week and enough peptide remains for many more conjugation reactions. By contrast 16 individual conjugate syntheses, RP-HPLC purifications for each peptide, and subsequent conjugates as well as conjugate desalting would take a minimum of 48 hours each, excluding method optimization.
Splicing redirection activity of LB1-PNA705 conjugate library
The PNA705 conjugates of LB1 were assessed for their ability to enter HeLa pLuc705 cells and redirect splicing, resulting in up-regulation of luciferase expression. Results were obtained with 48-well plates, which were used for all screening assays shown in this work. Several conjugates were purified by RP-HPLC and assayed, which provided similar results compared to the crude conjugates obtained by SELPEPCON (Figure 22).
The full LB1-PNA705 conjugate library was assayed at a single-concentration (5 μΜ) and the results analysed as the fold increase in luminescence compared to buffer only (Figure 2). Several of the LB1-PNA705 constructs were strongly active, as was expected for Arg- rich peptides. Substantial reduction in the number of Arg residues (e.g. multiple replacements by His, Ser or Glu) led to significantly lower activity (LB1_10 - 12). When the Arg residues were replaced by Lys, retaining the overall positive charge, some splicing redirection activity was retained (LB1_9 and 13 - 16). LB1-PNA705 conjugates differ slightly from CPP-PNA constructs previously synthesized by us.6' 10 Thus LB1-PNA conjugates contain an additional free Cys residue and the peptide to PNA conjugation is N- to -terminus rather than C- to /V-terminal conjugation used in previous work.
However, LB1_1 containing the original Pip5h sequence showed similar splicing redirection activity to that of previously synthesised Pip5h-PNA705 (data not shown). Amongst the more active conjugates, variations in splicing redirection activity observed in the HeLa cells were not identical to those that might have been expected based on knowledge of exon skipping effects in mouse mdx muscle cells for which the Pip5 and the later Pip6 series were designed.10 11 Scrambling of the hydrophobic core (LB1_3), replacement by Ala residues (LB1_4), substitution of Leu by Pro (LB1_8) or a negatively charged Glu (LB1_6) all appeared to be detrimental to the activity (Figure 2). Good splicing redirection activity was retained by reversal of the core sequence (LB1_2) or by replacement of a Leu by a Trp (LB1_7). The latter gives an IWFQ sequence also found in Penetratin, from which the hydrophobic core sequence of the Pip series was originally derived. Interestingly, the replacement of same Leu by a Lys residue significantly increased activity (LB1_5).
The results of CPP-PNA705 conjugate library based on LB1 demonstrate how
SELPEPCON can be effective for study and optimization of the amino acid sequence of a CPP for delivery of a cargo into a particular cell system and where a cellular assay is available.
Parallel synthesis and purification of LB2 CPP-library
We now wished to apply the SELPEPCON methodology to a significantly larger library in order to demonstrate its potential for selection of a CPP from a large range of sequence- dissimilar peptides, which would be necessary in the case of a new cargo candidate where relatively little was known about likely requirements for intracellular activity. Thus we designed peptide library LB2 consisting of 78 peptides that included many well-known CPPs, some sequences obtained from searching scientific literature, and some newly designed (Tables 3-5, Figures 12-14). The peptides in this library consisted of both hydrophobic and hydrophilic peptides, ranged in length from 6-28 amino acids and carried a net charge ranging from -1 to +13. The library consisted of standard L-amino acids with the addition of β-alanine (B) and ε-aminocaproic acid (X), which are commonly found in CPPs.¾ c Since it was found that free thiol groups inhibited the copper-catalysed cycloaddition reaction, Cys residues could not be introduced using Cys(Trt), which is the standard protecting group used in peptide synthesis. Instead an alternative procedure was developed using S-te t-butylthio-L-cysteine, which contains a disulfide-protecting group that is stable to TFA cleavage but which is released in the reducing step of the conjugate workup procedure.
The library of CPPs (LB2) was synthesised in parallel using the procedure described above for LB1. Only standard synthesis protocols were used for the parallel Synthesizer and a single method for purification and isolation was employed without any peptide specific optimization. Using these standard conditions, all but 5 of the 76 peptides (>90%) were successfully obtained, where the expected masses were seen as main signals in mass spectrometry (Tables 3 - 8 (Figures 12 - 17) and Figures 18(C) - 18(L)). Because of the type of work-up procedure, small amounts of impurities were seen in most of the mass-spectra obtained for the successfully synthesised peptides, but in 4 peptide syntheses a significant amount of shorter acetyl-capped peptide sequences were observed. Overall yields for the 71 successful syntheses were excellent (±70% average) and provided plenty of peptide material in principle for many different conjugation reactions on nanomole scale for this CPP library.
Since 20 peptides could be purified easily in parallel, the library was obtained in a total of only 9 days, including time for automated peptide assembly. Further parallelization to allow for the purification of more peptides simultaneously should lead to further decreases in this time.
LB2-PNA705 conjugate library synthesis
The LB2 peptides were conjugated to PNA705 using the procedure described previously for LB1 conjugations. Most conjugates were obtained in excellent yield (>60% average, Tables 3 to 8, Figures 12 to 17). As in the case of LB1 , a small amount of unconjugated PNA was observed by MALDI-TOF mass-spectroscopy and HPLC for all conjugates obtained (Figures 20(D) - 21(B)). Out of the 72 peptides where there was a peptide product available for conjugation, 10 conjugates were either not obtained or showed only small amounts of conjugate product compared to unconjugated PNA following affinity purification and desalting. The failed conjugations were practically all for the longer peptides that are likely to be more prone to form secondary structures, which may sterically inhibit the conjugation reactions. In the cases of all 3 peptides that showed significant amounts of acetyl-capped peptide impurities, good quality conjugate products were nevertheless obtained. This
demonstrates the effectiveness of the introduction of the alkyne linker in the final step of assembly in combination with acetic anhydride capping after each synthesis cycle during peptide synthesis, which allows for the removal of these peptide-impurities during affinity purification of the conjugates.
The four LB2-PNA705 conjugates that contained Cys residues (see Tables 3 to 8, Figures 12 to 17 and 20) showed by mass-spectrometry impurities that resulted from a statistical mixture of te/f-butylthio (StBu) modifications. Some modification was even observed of the Cys residue that remains attached to PNA705 after release from the affinity support, which was evidenced by the fact that conjugate masses were found corresponding to two StBu groups for conjugates that contained only one Cys residue in the peptide part. This may be because residual free StBu that is not effectively removed by the OASIS cartridge workup of the conjugates can slowly allow StBu disulfides to form with available Cys residues in the conjugates. The StBu disulfide protecting groups are clearly cleaved off during the reduction step in SELPEPCON, since the alternative use of centrifugal filter units (Amicon Ultra 3K 0.5 ml, Milipore Ireland Ltd) to remove salts, TCEP reducing agent and StBu did provide the main product conjugates without StBu modifications. However, this procedure resulted in low yields, especially for hydrophobic conjugates.
Once again the synthesis and purification procedures were very fast and effective. In addition to the good yields of the large majority of the conjugates, the conjugations and purifications involved in the whole library could be carried out within 8 days.
Splicing redirection activity of LB2-PNA705 conjugate library
The LB2-PNA705 conjugate library was screened at 5 μΜ concentration for their ability to enter HeLa pLuc705 cells and redirect splicing, resulting in up-regulation of luciferase expression (Figure 3). A significant number of active conjugates were identified from this initial screen, showing 20- to 200-fold increases in luminescence values. In some cases, high error-bars were observed for conjugates that showed over a 20-fold increase, because conjugates could not all be tested in a single 48-well plate experiment and samples with high luminescence readouts show large variability between different experiments. However, the main purpose of the luciferase readout is to distinguish active from inactive conjugates. As a check on the reliability of purifications of conjugates several conjugates were purified by HPLC and tested for their ability to up-regulate luciferase expression (Figure 4). None of the tested low-activity crude conjugates showed significantly higher activity after purification, revealing good reliability of the crude conjugates in this assay. The mildly active PNA705 conjugate of LB2_21 does show improved activity after purification. This can be explained because the crude conjugates contain a small amount (±10%) of unconjugated PNA, lowering the effective concentration of the LB2_21-PNA705. This highlights once again that the assay results using this crude library should only be used to find suitable active CPP-candidates for further investigation and active conjugates should not be directly compared.
Some interesting observations can be inferred from the results of the luciferase expression readouts. First, strong splicing redirection for the PNA705 cargo in this HeLa- cell assay is clearly directly related to the numbers of positive charges, particularly Arg residues, in the peptide part of the conjugates (Figure 5a and 5b). A charge of at least +8 and at least 7-8 arginines are revealed as good CPPs properties. Association between the number of Arg residues and cell penetrating ability for CPP conjugates of PNA and PMO cargoes is well known from previous literature12, but never before has such a large number and variety of CPPs of varying Arg content been tested in parallel. Interestingly several standard CPP sequences previously proposed for conjugation to PNA/PMO cargoes were found to be ineffective in this assay (for example, two containing Tat sequences LB2_30 and 52, and two containing Penetratin sequences LB2_28 and 35). These results show that effective cell penetration is cargo and cell dependent and demonstrates how SELPEPCON can be a valuable tool in the selection of a good CPP candidate for a particular application. An additional experiment was carried out in which all conjugates were evaluated in a single point assay to demonstrate how a first round of selection can be performed after a minimal screening effort (Figure 23). Although, several active conjugates give very high activity spikes, the same trend for active conjugates is observed as for the multiple data-point screening shown in Figure 3.
A follow-up to screening can provide further information on selected candidate
conjugates. For example, initial "hits" can be confirmed by evaluation of splicing redirection in addition at the RNA level.6 This assay is more reliable compared to the luciferase readout but is less suitable in a high-thoughput mode because of the need for gel electrophoresis to separate and quantify DNA strands. An example is shown in Figure 6 in which several LB2-PNA705 conjugates were assessed by extraction of RNA from the treated HeLa pLuc705 cells followed by RT-PCR. Previously reported Pip-1-PNA6, prepared by conventional conjugation through a C- to A erminai disulfide linkage was used as a positive control. As expected, the splice corrected RNA levels were low when cells were exposed to LB_54-PNA705, which also shows very low activity in the luciferase readout. LB2_7 contains the same Pip1 sequence as the positive control. The PNA705 conjugate was N- to A erminally linked as obtained through SELPEPCON and showed high splice correction activity at both 2.5 and 5 μΜ. At 5 μΜ fully spliced RNA levels are high for the PNA705 conjugates of LB2_30, 36 and 59, while LB2_30 showed significantly lower luciferase activity. The saturation of the splicing redirection observed at 5 μΜ can explain the large variation in the luciferase activity assay. At 2.5 μΜ these conjugates show the same activity trend by RT-PCR as seen in the luciferase readout obtained at 5 μΜ, highlighting the difference in sensitivity between the two methods. SELPEPCON assessment and application prospects
The SELPEPCON methodology described above has been exemplified by use of libraries of CPPs conjugated to a PNA705 cargo where a reliable cell assay is available to gauge the effectiveness of the peptides in delivering the cargo into the nucleus of HeLa cells. Clearly the methodology is in principle applicable to any bio-cargo capable of being functionalized by an azido group, such as PMO or other oligonucleotide types, siRNA, peptides or other biomolecules where there is a need to search for peptides that when conjugated to a cargo may enhance its delivery into cells or for cell or tissue targeting. For example, we are currently applying the SELPEPCON technology to cell delivery of a potential anti-cancer peptide known to poorly enter cells itself.
We chose to conjugate the \/-terminus of peptides to the N-terminus of the cargo, since N-terminal peptide functionalization is technically easier and in addition capping of unreacted chains during synthesis reduces the possibility of shorter chain peptide impurities becoming conjugated to the cargo. Thus, 3 less pure peptides could nevertheless be conjugated to the PNA cargo and still lead to an acceptable conjugate product. However, the method is not limited to A/-terminal conjugation via a A erminal alkyne. To demonstrate this we synthesized two peptides based on LB1 containing C- terminal alkynes (LB1_17 and LB1_18, Figures 24 and 25 (Tables 9 and 10)). The alkyne was placed on the C-terminus by use of A/-Fmoc- L-bishomopropargylglycine-OH (Fmoc- Bpg-OH, Chiralix, Nijmegen, The Netherlands) during solid-phase peptide synthesis. Both peptides were obtained in good yield. The double glycine spacer of LB1_18 did not appear to be necessary to improve the conversion in the conjugation reaction, since both conjugates were readily obtained in excellent yield and purity.
Following initial selection and verification of hits by the SELEPEPCON methodology using a wide CPP library, such as LB2 described above, lead optimisation would likely involve further rounds of SELPEPCON screening using narrower peptide libraries, as exemplified in LB1. Because the peptides in LB1 are relatively similar, conjugation efficiency and thus effective concentration and purity of the conjugates obtained by SELPEPCON will be less variable compared to a broad library such as LB2.
The resultant libraries are also suitable for other screening purposes such as
comparisons of serum stabilities of the conjugates, if in vivo studies are to be
contemplated. Once one or more candidate peptides have been chosen for the desired cargo, thereafter drug development might consist of investigation of alternative conjugation methods to vary the type of linkage between peptide and cargo, as well in some cases their respective orientations to each other. Commonly such studies are best carried out in an in vivo model of a disease state using conjugates prepared more conventionally on larger scale6' 9 12a- 3, since pharmacological properties of conjugates may vary substantially depending on cargo type as well as on peptide sequence. It is possible that SELPEPCON could be used for preparing conjugates for assay by intramuscular injection (e.g. into a mouse mdx model of Duchenne muscular dystrophy6) where only pg quantities of conjugates are needed. However, use of SELPEPCON should reduce considerably the time and effort required to obtain potential leads that are demonstrated to be capable of the required biological effect in a cell model, hitherto a major bottleneck.
We have developed a novel rapid parallel synthesis methodology (SELPEPCON) for the synthesis of libraries of conjugates of peptides with a PNA cargo and assayed the conjugates in a splicing redirection assay in HeLa pLuc705 cells. Alkyne and azide functional groups, which are known to be compatible with the most commonly used bio- molecules, are readily introduced on peptide and cargo moieties respectively. The methodology is capable of robotization and is applicable to large libraries of peptide conjugates of biomolecules, such as oligonucleotides and their analogues, peptides, siRNA etc., and in amounts suitable for in vitro or cell screening. We expect the
SELEPEPCON method to have wide applicability in drug discovery. Example 2 - SELPEPCON using peptide cargos
In order to further demonstrate the use of SELPEPCON we looked to apply the methodology to a peptide cargo of interest. Small peptides that inhibit the interaction between P53 and MDM2 were considered suitable peptide cargos for this purpose.1 P53 is a protein that is involved in tumour suppression and is compromised in many cancers because of MDM2 up-regulation. This makes inhibition of the P53-MDM2 interaction of particular interest for cancer treatment. Several very potent peptide sequences have been identified using in vitro screens and their effectiveness has been demonstrated in vivo.2 However, efficient cell-delivery has not been achieved thus far. SELPEPCON can provide a whole range of CPP-conjugates of these inhibitors that can be screened to find a suitable construct for efficient delivery. Application of SELPEPCON is described below.
Cargo synthesis
In order to apply SELPEPCON to peptide inhibitors of the P53-MDM2 interaction and in order to apply previously synthesized CPP-libraries LB1 and LB2, one potent peptide inhibitor needs to be modified with an azide functionality and a biotin tag. The PMI (TSFAEYWNLLSP) was selected as target cargo29. PMI and as control, P53
(ETFSDLWKLLPE) which is the original MDM2-binding site of 17 28P53, and these sequences with R4-tags on C- and N-termini were all synthesized. In addition, constructs were synthesized containing an N-terminal R4 group and a C-terminal Cys residue to represent as closely as possible an actual construct that would be obtained using SELPEPCON. The peptides were tested for their ability to inhibit the P53-MDM2 inhibition using a fluorescence polarization assay, as set-up by Fersht and colleagues (Figure 26, Table 11 ).3 The data confirmed that PMI binds much more strongly compared to the native 17-28P53 binding site. Additionally, it revealed that the modification of the peptide on either the N- or the C-terminus did not affect the ability of the sequence to bind MDM2. This is important since this does suggest that conjugation to PMI should in principle not affect the potency of this peptide.
The next step for the application of PMI in SELPEPCON was to produce a
difunctionalized version of PMI containing an azide and biotin tag linked via a disulphide bridge. One hurdle for the application of PMI was the poor water-solubility of this peptide. In order to avoid solubility issues upon conjugation of hydrophobic CPPs we attached a short spacer (GNGKKGG) to improve water solubility. The sequence
GNGKKGTSFAEYWNLLSPC (solPMI-SH) was synthesized on solid support by solid phase synthesis and modified at the N-terminal amino group using 5-azido pentanoic acid. The azide modified PMI (N3-solPMI-SH) was then modified with a biotin function using EZ-link HPDP-Biotin (Thermo Scientific). The product (N3-solPMI-S-S-biotin) was purified by HPLC and used for conjugation reactions. Additionally, N3-solPMI-S-StBu was synthesized in order to perform several test click-conjugations in which the product was be isolated by HPLC-purification.
Conjugations
Several peptides from LB2 were conjugated using the SELPEPCON conditions to N3- solPMI-S-StBu and the product was isolated by HPLC (Figure 27, Table 12). The conversion was high and the products were obtained in good yield. A separate selection of peptides from the LB2 CPP library was used for conjugation to N3-solPMI-S-S-biotin and the SELPEPCON procedure was applied for isolation and purification of the conjugates. Conjugation reactions all went to completion and the products were obtained in excellent yields with the exception of LB2_53-solPMI-S-S-biotin that obtained in a disappointing 18% yield. This was the most hydrophobic peptide of the selection and we therefore assume that most of the product was lost in one of the workup steps due to low water solubility or significant hydrophobic interaction with the OASIS cartridge. Overall the good conversion and yields demonstrate the general applicability of the SELPEPCON method at least to peptide based cargos.
Assay
The obtained constructs were used in a cell-based assay. A p53 reporter plasmid containing 14 copies of a p53 enhancer element sequence upstream of the luciferase gene was transfected into several cell-lines and the luciferase readout was determined using Nutlin-3, which is a small-molecule cell-permeable inhibitor of the MDM2-P53 interaction with a Kd similar to that of PMI in vitro. A good response was found in MCF-7 cells, however it was found that most of the conjugates appeared to be toxic at concentrations of 5 μΜ and above. Assaying the peptides at 1 and 2 μΜ a small dose response was observed.
General
MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro
BioSpectrometry workstation. A stock solution of 10 mg mL"1 of
a-cyano-4-hydroxycinnamic acid in 60% acetonitrile in water was used as matrix. Error bars are ± 0.1 %. Reversed phase HPLC purifications and analyses were carried out using a Varian 940-LC. A Nanodrop 2000 UV analyser (Thermo Scientific) was used for the quantification of peptide-cargo and peptide-cargo conjugate concentrations using the absorption at 280 nm with the following values to calculate absorption coefficients; W = 5.5 cm-1M-\ Y = 1.49 οητ Μ \ disulphide-bridge = 0.1 1 crrr1M-1. TBTA (tris- (benzyltriazolylmethyl)amine) was synthesised according to a literature procedure.4 Peptide synthesis was carried out on a 5 pmol scale using an Intavis Parallel Peptide Synthesizer, applying standard Fmoc chemistry and following manufacturer's
recommendations. The solid support was as supplied by Intavis (Tentagel, 0.2 mmol g~1). Double coupling steps were used with a PyBop/NMM coupling mixture followed by acetic anhydride capping after each coupling step.
N3-S0IPMI-SH synthesis
The PMI sequence23 containing a C-terminal C and an N-terminal G-N-G-K-K-G sequence for solubility (total sequence: NH2-GNGKKGGTSFAEYWNLLSPC-CONH2) was synthesised on solid support (see above). Terminal azide functionalization involved a manual coupling procedure using 5-azido penanoic acid and standard coupling reagents. The peptide was cleaved from the support and deprotected by addition of TFA (1.5 ml_) containing 5% triisopropylsilane/2.5% water and 1 % phenol with shaking for 2 hours. The mixture was filtered and concentrated. The crude PNA was isolated by cold diethyl ether precipitation, dissolved in water, filtered and purified by HPLC. A Phenomenex Semiprep C18 Jupiter column (250 x 10.00 mm, 5 micron) was used with the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 20% B 2-25 min 20%-60% B 25-30 min 60%-90% B (retention time: 18.1 min). The fractions containing the compound were combined and freeze-dried giving a fluffy white solid (yield 48% based on support loading). Mass, expected: m/z 2255 found: m/z 2253.
N3-soIPMI-S-S-biotin construct
To a solution of 860 nmol N3-S0IPMI-SH in 1 ml water, was added 2 mol
EZ-link HPDP-Biotin (Thermo Scientific) from a 3.7 pmol ml"1 stock in DMSO. To this solution 00 μΙ 2M sodium acetate pH 7 was added and the resulting solution shaken for two hours. The reaction was quenched by the addition of 1 ml 0.1 % TFA. This solution was filtered and then purified by HPLC using a Phenomenex Semiprep C18 Jupiter column (250 x 10.00 mm, 5 micron) was used with the following gradient (A: 0.1 % TFA, B: 90% acetonitrile, 0.1 % TFA) 0-2 min 20% B 2-25 min 20%-60% B 25-30 min 60%-90% B (retention time: 21 .6 min). The purified N3-solPMI-S-S-biotin was obtained as a fluffy white powder in ±20% yield. Mass, expected: m/z 2688 found: m/z 2681. Peptide library synthesis
Alkyne modified CPP-peptides were taken from library LB2 used for the synthesis of PNA705 conjugates.
Conjugate synthesis
A mixture was prepared containing 30 nmol N3-solPMI-S-S-biotin from a stock solution in water (±2 mM), 150 nmol alkyne modified peptide from a stock solution in NMP (± 10 mM), 0.2 μΙ 2,6-lutidine (1.7 μηιοΙ) and 1 μΙ diisopropylethylamine (5.7 μηηοΙ). 7.5 μΙ of 20 mM CuSCVTBTA solution (150 nmol) premixed in a 1 :1 mixture of DMSO and 10 μΙ of a 20 mM solution of sodium ascorbate (200 nmol) were added to this mixture. The mixture was left for one hour and quenched with 100 μΙ 0.2 M EDTA 40% acetonitrile and 800 μΙ TBS 40% acetonitrile. The solution obtained was vortexed and loaded in two batches of 500 μΙ on a streptavidin HP SpinTrap (GE Healthcare UK Limited) and each batch was incubated for 20 minutes while being mixed by inversion. The column was washed with 400 μΙ 0.1 M EDTA in TBS 40% acetonitrile and with 5x 400 μΙ TBS 40% MeCN. The conjugate was released from the resin by 2x 20 minutes reactions with 2x 400 μΙ 10 mM TCEP in TBS 40% acetonitrile and the solid support washed with 200 μΙ 40% acetonitrile in TBS. The solutions collected were combined and freeze-dried. The solid was dissolved in 500 μΙ 20% acetonitrile and loaded on an equilibrated 1 cc Oasis HLB cartridge
(Waters) together with 500 μΙ 0.1 % TFA. The column was washed with 3x 1 ml 0.1 % TFA, 3x 1 ml 5% acetonitrile 0.1% TFA and 1x 1 ml 0% acetonitrile 0.1 % TFA. These extensive washes are required to remove the TCEP from the cartridge. The conjugates are released using 500 μΙ 60% acetonitrile and diluted with 50 μΙ 0.1 % TFA. The resulting solution was freeze-dried and dissolved in 20-100 μΙ water based on the calculated molar absorption of the conjugates at 280 nm (see above). The concentration was determined and the solution was stored in the freezer for use in cellular assays (Yields see Figure 27, Table 12). P53-MDM2 inhibition assay
The assay is carried out in 48-well plated MCF-7 cells at 50-60 % confluency. First, 250 mg per well of p53-Luc plasmid is transfected by lipofection with Lipofectamine2000. Then the next day cells are incubated for 4 h with CPP-PMI conjugates in OptiMEM, followed by addition of standard media of DMEM/10% FBS and further incubation for 20 h. Next day media is changed for the fresh DMEM/105FBS and cells are left for another 24h incubation followed by Luciferase assay according to standard protocol. Example 3 - SELPEPCON for a PMO cargo
Method 1. Peptide-PMO conjugate synthesis using doubly modified PMO
In order to demonstrate how SELPEPCON can be applied to PMO we synthesized a bifunctional PMO cargo containing a disulphide bridge biotin and an azide functionality. In order to achieve this we acquired a commercial 25-mer exon skipping PMO containing a disulphide and a free amine and modified this following the synthesis steps outlined in Figure 28.
To test the SELPEPCON methodology one example of conjugation was performed to LB2_9 of the previously reported peptide library. Because of the value of the PMO material, the scale was reduced to 3 nmol. Nevertheless, using the standard volumes and cartridge sizes for SELPEPCON at 30 nmol, the LB2_9-PMO-SH conjugate was obtained at good yield and purity. This demonstrates that SELPEPCON can be applied to other oligonucleotide analogue based cargos such as PMO. a further method was developed to show the applicability of PMO as a cargo. Both methods have the advantage that the starting PMO is unmodified and does not contain either a disulphide on one end or a primary amine modifier on the other end. In each case a single peptide, LB2_9 was chosen to exemplify this method, but it should be noted that these synthesis methods can also be applied to a plurality of peptides in parallel.
Method 2. Peptide-PMO conjugation via amide bond formation
In this method of SELPEPCON application to a PMO cargo (Figure 3 ), the use of Click conjugation (azido to alkyne) was replaced by amide bond formation effected by pre- activation of the carboxylic acid function of the peptide component. Thus LB2_9 peptide was first synthesized N-terminally acetylated and with an additional three-amino acid CXB C-terminal extension where the C-terminus is a free carboxylic acid. The peptide product (LB2_9-SH) was then biotinylated in DMSO/ sodium phosphate buffer solution using EZ- link HPDP-Biotin for 2 h at room temperature and the product purified using reversed- phase HPLC.
The biotinylated LB2_9_SH was conjugated to the 3'-end of the PMO through its C- terminal carboxyl group by reaction with HBTU/HOAt/DIPEA in NMP for 2 h at room temperature. The resulting solution was immobilized on a streptavidin HP SpinTrap column and the conjugate was released by reaction with TCEP. The resulting solution was purified on an Oasis HLB cartridge to yield the desired conjugate. The free thiol group in the conjugate was capped by treatment with iodoacetamide in sodium bicarbonate solution for 2 h in the dark and excess iodoacetamide was removed through the use of an Amicon® Ultra-1 ml_ centrifugal filter unit as before.
General
MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro
BioSpectrometry workstation. A stock solution of 10 mg mL1 of sinapinic acid in 60% acetonitrile in water was used as matrix. Error bars are ± 0.1 %. Reversed phase HPLC purifications and analyses were carried out using a Varian 940-LC. A Nanodrop 2000 UV analyser (Thermo Scientific) was used for the quantification of PMO concentrations.
Bifunctional PMO (3'-disulfide, 5' primary amine) as well as unmodified PMO were purchased from Gene Tools LCC, Philomath, OR, USA. The PMO sequence is 5' to 3' GGCCAAACCTCGGCTTACCTGAAAT. For determination of the concentration Optical Density was measured at 265 nm using a molar absorption coefficient of 259.21 cnr1M~1 as provided by the supplier. EZ-link HPDP Biotin was bought from Thermo Scientific, Waltham, MA, USA. Method 1
Synthesis of HS-PMO-NH2
The PMO (3'-disulfide, 5' primary amine) was dissolved in sterile water (around 1 nmol/μΙ, 1 mM). The concentration of the solution and the amount of PMO was determined and 1/5th of the same volume of 10 mM TCEP in TBS was added, and the solution mixed for 30 min. If there are any particles, the solution was filtered using a SPIN-X column. The solution was loaded in an Amicon® Ultra (Merck, Millipore, Darmstadt, Germany) 0.5 ml centrifugal filter and the solution concentrated and washed several times (5x) with water. The washed HS-S-PMO-NH2 was transferred to a new tube and lyophilized. Mass, expected: m/z 8780 found: m/z 8788.
Synthesis of Biotin-S-S-PMO-NH2
The obtained HS-S-PMO-NH2 was dissolved in water (around 1 nmol/μΙ) and the concentration of the solution and the amount of PMO was determined. 3 equivalents of EZ-link HPDP Biotin (Thermo Scientific) from an 18.5 mM (10mg/ml) solution in DMSO was added and enough 2M NaOAc pH 7 to make a 200 mM NaOAc. The solution was mixed for 30 minutes. The solution was diluted with water and filtered using a SPIN-X column (Corning Incorporated, Corning, NY, USA) and loaded on HPLC (Phenomenex C18 Jupiter column 10-60% B in 30 min). Collected fractions were lyophilized. Mass, expected: m/z 9216 found: m/z 9223. Synthesis of Biotin-S-S-PMO-N3
PMO was dissolved in DMSO to give a solution of about 10 mM. In a separate vial were mixed 2.5 eq 5-azidopentanoic acid in NMP (100 mM), 5.75 eq HBTU in NMP (300 mM), 5 eq HOAt in NMP (300 mM) and 5.75 eq DIPEA (neat). After the addition of DIPEA this mixture was added to the PMO solution and heated for 2 hours at 37°C. The reaction was quenched by addition of a four-fold volumetric excess of 0.1 % TFA in water (minimum 200 μΙ) and the resulting mixture filtered using a SPIN-X column (Corning Incorporated, Corning, NY, USA). The filtered solution was purified on HPLC (Phenomenex C18 Jupiter column 10-60% B in 30 min). Collected fractions were lyophilized. Mass, expected: m/z 9342 found: m/z 9350.
Conjugation of alkyne peptide LB2_9 to biotin-S-S-PMO-N3
Standard SELPEPCON conditions were used for the conjugation and workup to obtain the LB2_9-PMO-SH conjugate from alkyne functionalised LB2_9 (Table 3, Figure 12) and Biotin-S-S-PMO-N3. The only difference was that only 3 nmol of cargo was used. All the volumes of the cartridges and the washings were kept the same. The LB2_9-PMO-SH conjugate was obtained in 1.6 nmol (55%) yield. Mass, expected: m/z 1 1895 found: m/z 1 1870.
Method 2
Alternative method of conjugate synthesis without a connector peptide LB2_9 -SH peptide synthesis
LB2_9 peptide (Table 3, Figure 12) was synthesized N-terminally acetylated and with an additional three-amino acid CXB C-terminal extension where the C-terminus is a free carboxylic acid (Ac-RXRRBRRXRYQFLI RXRBRXRCXB where B is β-alanine and X is aminohexanoic acid) on an Fmoc-p-Ala-Wang resin ( 00-200 mesh, 0.6 mmol/g) using Fmoc chemistry on a CEM Liberty™ microwave peptide synthesizer (Buckingham, UK). The side chain protecting groups used were trifluoroacetic acid labile. The peptide was synthesized on a 0.1 mmol scale using a 5-fold excess of Fmoc-protected amino acids (0.5 mmol), which were activated using PyBOP (5-fold excess) in the presence of DIPEA. Piperidine (20% v/v in DMF) was used to remove N-Fmoc protecting groups. The coupling was carried out once at 75 °C for 5 min at 60-watt microwave power except for arginine residues, which were coupled twice each. Each deprotection reaction was carried out at 75 °C twice, once for 30 sec and then for 3 min at 35-watt microwave power. Once synthesis was complete, the resin was washed with DMF (3 x 50 ml_) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA. The peptide was cleaved from the solid support by treatment with a cleavage cocktail consisting of trifluoroacetic acid (TFA): 3,6-dioxa-1 ,8-octanedithiol (DODT): H20: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1 %, 10 mL) for 3 h at room temperature. Excess TFA was removed by blowing N2 through the peptide solution. The cleaved peptide was precipitated via the addition of ice-cold diethyl ether and centrifuged at 3000 rpm for 2 min. The peptide pellet was washed in ice-cold diethyl ether thrice. The crude peptide was dissolved in water, analyzed and purified by RP-HPLC on Phenomenex Jupiter columns (4.6 x 250 mm, C18, 5 μΐτι) and (21.2 X 250 MM, C18, 10 μη-ι) respectively. A flow rate of 1 .5 mL/min for the analytical column and 0 mL/min for the preparative column with the following gradient (A: 0.1 % TFA, B: 90% CH3CN, 0.1 % TFA) 0-2 min 5% B 2-35 min 5%-60% B 35-40 min 60%-90% B was used. The fractions containing the desired peptide were combined and lyophilized to give the product as a white solid (55% yield). Mass, expected: m/z 3159 found: m/z 3166. Biotinylation of LB2_9-SH
To a solution of (2 mmol) of LB2_9-SH in DMSO (66 mL) was added water (200 mL). To this solution EZ-link HPDP-Biotin (267 mL of 15 mM in DMSO) and sodium phosphate buffer pH 7.4 (66 mL of a 100 mM solution) were added and the resulting mixture was left for 2 h at room temperature. The reaction was quenched by the addition of 0.1 % TFA (0.66 mL). This solution was filtered and the desired product was isolated using RP- HPLC. A Phenomenex Jupiter column (21.2 χ 250 mm, C18, 10 mm) was used at a flow rate 10 mL/min with the following gradient (A: 0.1 % TFA, B: 90% CH3CN, 0.1 % TFA) 0-30 min 10%-70% B 30-33 min 70%-90% B. The fractions containing the desired peptide were combined and freeze dried to yield the biotin-modified peptide as a white solid (48% yield). Mass, expected: m/z 3588 found: m/z 3595.
Conjugation of biotinylated LB2_9-SH to PMO and capping
The biotinylated LB2_9-SH was conjugated to the 3'-end of the 25-mer PMO antisense sequence targeting mouse dystrophin exon-23, through its C-terminal carboxyl group.
This was achieved using a 2.5 and 2-fold excess of HBTU and HOAt in NMP respectively in the presence of 2.5 eq of DIPEA and a 2-fold excess of peptide over PMO dissolved in DMSO was used. The reaction was carried out at 40 °C for 2 h and quenched with EDTA (900 mL of 10 mM in TBS 10% CH3CN). The resulting solution was loaded in two batches of 500 mL on a streptavidin HP SpinTrap (GE Healthcare) and incubated for 1 h. The column was washed with EDTA (400 mL of 10 mM in TBS) and with TBS (5 400 mL).
The conjugate was released from the resin by 2 χ 20 min reaction with TCEP (2 400 mL of 10 mM in 20% CH3CN TBS) and the resin washed with TBS (200 mL). The resulting solutions were combined and lyophilized. The resultant white solid was dissolved in 0.1 % TFA 10% CH3CN (500 mL) and loaded on to an equilibrated 1 cc Oasis HLB cartridge (Waters) together with 0.1% TFA (500 mL). The column was washed with 0.1 % TFA (3 χ 1 mL), 5% CH3CN in 0.1 % TFA (3 1 mL) and 10% CH3CN in 0.1 % TFA (1 1 mL). The conjugate was released from the cartridge via the addition of 60% CH3CN in 0.1 % TFA (500 mL). The resulting solution was lyophilized to yield the desired conjugate as a white solid (42 % yield). Mass, expected: m/z 1 1564 found: m/z 1 1583.
The conjugate (10 nmol) was dissolved in sodium bicarbonate solution (25 mL of 0.1 M, pH 8.0) and iodoacetamide (23 mg, 250 nmol) was added. The solution was left for 2 h in the dark and diluted with water (975 mL). Excess iodoacetamide was removed through the use of an Amicon® Ultra-1 mL centrifugal filter unit with a 3000 molecular weight cut off. The resulting filtrate was lyophilized to yield the desired S-capped product (55%). Mass, expected: m/z 1 1622 found: m/z 1 1642.
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Claims

Claims:
1. A method of forming a peptide-cargo conjugate, the conjugate comprising a peptide bonded to a cargo molecule, the method comprising:
(i) contacting a peptide attached to a first functional group at the N- or C- terminus with a cargo construct comprising a cargo molecule attached to a second functional group, the cargo molecule also comprising a tag, under conditions that allow reaction between the functional groups to form a bond between the peptide and cargo molecule, thereby forming a peptide- cargo construct conjugate in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(ii) partitioning the peptide-cargo construct conjugate from unreacted peptide by binding the tag to a capture element;
(iii) releasing the peptide-cargo conjugate from the tag.
2. A method of forming peptide-cargo conjugates, the conjugates each comprising a peptide bonded to a cargo molecule, the method comprising:
(a) preparing a plurality of peptides each attached to a first functional group at the N- or C- terminus;
(b) preparing a plurality of cargo constructs each comprising a cargo molecule
attached to a second functional group, the cargo molecule also comprising a tag;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags.
3. A method of forming a peptide-cargo conjugate, the conjugate comprising a peptide bonded to a cargo molecule, the method comprising:
(i) contacting a peptide attached to a first functional group at the N- or C- terminus with a cargo construct comprising a cargo molecule attached to a second functional group, the peptide molecule also comprising a tag, under conditions that allow reaction between the functional groups to form a bond between the peptide and cargo molecule, thereby forming a peptide-cargo construct conjugate in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(ii) partitioning the peptide-cargo construct conjugate from unreacted peptide by binding the tag to a capture element;
(iii) releasing the peptide-cargo conjugate from the tag.
4. A method of forming peptide-cargo conjugates, the conjugates each comprising a peptide bonded to a cargo molecule, the method comprising:
(a) preparing a plurality of peptides each attached to a first functional group at the N- or C- terminus, the peptide molecule also comprising a tag;
(b) preparing a plurality of cargo constructs each comprising a cargo molecule
attached to a second functional group;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags.
5. The method of any one of claims 1 to 4, wherein the peptide has a maximum length of 60 amino acids and a minimum length of 2 amino acids.
6. The method of any one of claim 1 to 4, wherein the peptide has a length selected from the group consisting of: 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 29 amino acids, 5 to 28 amino acids, 5 to 27 amino acids, 5 to 26 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 22 amino acids, 5 to 21 amino acids, or 5 to 20 amino acids.
7. The method of any one of claims 1 to 6, wherein the conjugates comprise a peptide covalently bonded to the cargo molecule.
8. The method of any one of claims 1 to 7, wherein the first and second functional groups form a pair having selective reactivity towards each other.
9. The method of any one of claims 1 to 8, wherein the first and second functional groups are selected from one of: an azide and an alkyne; a thiol and an alkene; a thiol and an alkyne; a diene and a dienophile; an isonitrile and a tetrazine; an epoxy and an aziridine; an amine and an isocyanate, an active methylene and an activated olefin, a thioester and a cysteine thiol, an aldehyde and a hydrazine, an aldehyde and an aminooxy derivative.
10. The method of any one of claims 1 to 9, wherein the first functional group is attached to the terminal amine of the peptide.
11. The method of any one of claims 1 to 10, wherein the cargo molecule is an oligonucleotide, an oligonucleotide analogue, a peptide or a peptide analogue.
12. The method of any one of claims 1 to 10, wherein the cargo molecule is a peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), or siRNA.
13. A method of generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus; (b) preparing a plurality of cargo constructs of the same type, each comprising a cargo molecule attached to a second functional group, the cargo molecule also comprising a tag;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element; (e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
14. A method of generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus, the peptide molecule also comprising a tag;
(b) preparing a plurality of cargo constructs of the same type, each comprising a cargo molecule attached to a second functional group;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
15. A method of screening a plurality of peptide-cargo conjugates for a functional property of the peptide-cargo conjugate, the method comprising simultaneously assaying a plurality of different peptide-cargo conjugates for said functional property.
16. The method of claim 15, wherein the different peptide-cargo conjugates each comprise the same cargo molecule and comprise different peptides.
17. The method of claim 15, wherein the different peptide-cargo conjugates each comprise the same peptide and comprise different cargo molecules.
18. The method of any one of claims 15 to 17, further comprising providing a library of different peptide-cargo conjugates on which to perform the assay.
19. The method of any one of claims 15 to 18 further comprising generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising:
(a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus;
(b) preparing a plurality of cargo constructs of the same type, each comprising a cargo molecule attached to a second functional group, the cargo molecule also comprising a tag;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
20. The method of any one of claims 15 to 18 further comprising generating a library of peptide-cargo conjugates, the library having a plurality of different peptide-cargo conjugates, the method comprising performing a plurality of peptide-cargo conjugate synthesis reactions in parallel each synthesis reaction comprising: (a) preparing a plurality of peptides of the same type, each peptide attached to a first functional group at the N- or C- terminus, the peptide molecule also comprising a tag;
(b) preparing a plurality of cargo constructs of the same type, each comprising a
cargo molecule attached to a second functional group;
(c) contacting said peptides with said cargo constructs under conditions that allow reaction between the functional groups to form a bond between a respective peptide and cargo molecule, thereby forming a plurality of peptide-cargo construct conjugates in which the cargo molecule is bonded to the N- or C- terminus of the peptide;
(d) exposing the peptide-cargo construct conjugates to a plurality of capture elements allowing a tag to associate with a capture element;
(e) releasing peptide-cargo conjugates from their respective tags; wherein the peptides of (a) and/or cargo molecules of (b) are different between respective synthesis reactions, thereby generating a library of different peptide-cargo conjugates.
21 . A library of peptide-cargo conjugates, the library comprising a plurality of containers each containing a quantity of an isolated or substantially purified peptide-cargo conjugate.
22. A peptide-cargo construct conjugate comprising a peptide bonded to a cargo construct comprising a cargo molecule and a releasable tag, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide.
23. A plurality of peptide-cargo construct conjugates immobilised on a solid support through association of a tag with a capture element, wherein each peptide-cargo construct conjugate comprises a peptide bonded to a cargo construct comprising a cargo molecule and a tag capable of association with a capture element on the solid support, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide molecule.
24. The peptide-cargo construct conjugate of claim 22 or plurality of peptide-cargo construct conjugates of claim 23 wherein the cargo construct comprises a cleavable linker attached to the tag and to the cargo molecule, the cleavable linker linking the tag and cargo molecule.
25. A peptide-cargo construct conjugate comprising a peptide having a releasable tag, the peptide bonded to a cargo construct comprising a cargo molecule, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide.
26. A plurality of peptide-cargo construct conjugates immobilised on a solid support through association of a tag with a capture element, wherein each peptide-cargo construct conjugate comprises a peptide having a tag capable of association with a capture element on the solid support, the peptide bonded to a cargo construct comprising a cargo molecule, wherein the peptide is bonded to the cargo molecule at the N- or C- terminus of the peptide molecule.
27. The peptide-cargo construct conjugate of claim 25 or plurality of peptide-cargo construct conjugates of claim 26 wherein the peptide comprises a cleavable linker attached to the tag and to the peptide molecule, the cleavable linker linking the tag and peptide.
28. The library of peptide-cargo conjugates, peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates of any of claims 21 to 27, wherein the peptide has a maximum length of 60 amino acids and a minimum length of 2 amino acids.
29. The library of peptide-cargo conjugates, peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates of any of claims 21 to 28, wherein the peptide has a length selected from the group consisting of: 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 29 amino acids, 5 to 28 amino acids, 5 to 27 amino acids, 5 to 26 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 22 amino acids, 5 to 21 amino acids, or 5 to 20 amino acids.
30. The library of peptide-cargo conjugates, peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates of any of claims 21 to 29, wherein the conjugates comprise a peptide covalently bonded to the cargo molecule.
31. The library of peptide-cargo conjugates, peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates of any of claims 21 to 30, wherein the cargo molecule is an oligonucleotide, an oligonucleotide analogue, a peptide or a peptide analogue.
32. The library of peptide-cargo conjugates, peptide-cargo construct conjugate or plurality of peptide-cargo construct conjugates of any of claims 21 to 31 , wherein the cargo molecule is a peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO), locked nucleic acid (LNA), or siRNA.
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