Modified proteins and methods of manufacture thereof

Abstract

The present invention provides modified proteins and methods of manufacture of modified proteins. Also provided herein are compositions comprising such modified proteins and the use of those compositions in pharmaceutical applications.

Description

[0001] MODIFIED PROTEINS AND METHODS OF MANUFACTURE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention provides modified proteins, methods of producing modified proteins, and formulations and compositions comprising the modified proteins.

[0004] BACKGROUND

[0005] Nitric Oxide (NO) is a gaseous signalling molecule and plays a key role in neurotransmission, or information sharing between neurons, which helps functions in the nervous system like digestion and memory. NO may also encourage the release of hormones, including growth hormones and insulin. NO also acts as a vasodilator, meaning it helps open blood vessels to improve blood flow and may also improve immunity by promoting inflammation to fight infections.

[0006] Collagen is the major protein of the extracellular matrix (ECM) and is the most abundant protein found in mammals, comprising 25% of the total protein content and 70% to 80% of skin (dry weight). The central feature of all collagen molecules is their stiff, triplestranded helical structure. Types I, II, and III are the main types of collagen found in connective tissue and constitute 90% of all collagen in the body.

[0007] Previously, collagens were thought to function only as structural support; however, it is now evident that collagen and collagen-derived fragments control many cellular functions, including cell shape and differentiation, migration, and synthesis of a number of proteins. Findings suggest that cell contact with precise extracellular matrix molecules influences cell behaviour by regulating the quantity and quality of matrix deposition.

[0008] Wound healing is a complex process that involves coordinated interactions between diverse immunological and biological systems. Long-term wounds remain a challenging clinical problem, affecting approximately 6 million patients per year, with a high economic impact.

[0009] Wound healing is a process whereby the skin (or another organ-tissue) repairs itself after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) exist in a steady-state equilibrium and shielded from the external environment. When the skin is broken, the normal (physiologic) process of wound healing begins. The classic model of wound healing comprises three or four sequential, yet overlapping, phases:

[0010] Phase 1: Haemostasis.

[0011] Phase 2: Inflammation.

[0012] Phase 3: Proliferation.

[0013] Phase 4: Remodelling.

[0014] Epidermal stem cells (ESCs) are found in the dermis and have been shown to play an important role in skin homeostasis and wound healing. Upon skin injury, ESCs are recruited by the epidermis and migrate towards the centre of the wound. NO has emerged as a critical molecule for wound healing. It is a volatile gas produced in many tissues and organs of the body and acts as a gaseous messenger. During wound healing NO is released by various types of immune and skin cells and it has been found to have multiple biological effects including:

[0015] - Promoting cell migration and proliferation.

[0016] - Vasodilation.

[0017] - Stimulation of angiogenesis.

[0018] - Regulation of immune responses.

[0019] - Potent anti-microbial activity.

[0020] After skin damage, NO levels peak during the inflammation phase of wound healing, approximately one day after the injury, and gradually decrease as the healing process progresses.

[0021] Zhan et al., 2016 ("Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling", Scientific Reports, 2016; 6, 30687) incorporated herein by refrence indicates that an NO donor, S-nitroso-N-acetylpenicillamine (SNAP) facilitated the in vitro migration of human epidermal stem cells (huESCs) in both live-imaging and scratch models.

[0022] Han et al., 2012 ("Nitric Oxide-Releasing Nanoparticles Accelerate Wound Healing by Promoting Fibroblast Migration and Collagen Deposition" , The American Journal of Pathology. 2012; Vol. 180, No. 4, April) incorporated herein by reference describes the effects of NO-releasing nanoparticles on wound healing in mice and indicated that NO- nanoparticles (NO-np) were able to accelerate wound healing.

[0023] WO 2016 / 16166524 incorporated herein by reference relates to modified collagen comprising S-nitroso groups and a method of manufacture of such a modified collagen.

[0024] However, the clinical use of NO to date has been minimal, mainly due to the technical challenge of delivering it effectively to target tissues and also being able to store NO- containing material in a stable form prior to use. Additional technical challenges relating to the use of NO include ensuring release of the molecule from a medical device at a rate that is suitable for its intended clinical use.

[0025] The present invention seeks to meet the problems outlined above through the development of an effective and scalable method for producing modified proteins that are able to deliver exogenous NO (for example, a modified collagen, such as a modified collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI or XVII capable of delivering exogenous NO).

[0026] The modified proteins of the invention advantageously provide localised and controlled release of NO which can assist the body's natural biological response for a variety of applications, for example in wound healing. Furthermore, the modified proteins of the invention advantageously exhibit improved stability and are capable of delivering exogenous NO after storage and over an extended period of time. SUMMARY OF THE INVENTION

[0027] In a first aspect of the invention, there is provided a method of producing a modified protein, the modified protein comprising one or more groups of Formula I wherein :

[0028] X1represents the peptide chain of the modified protein; each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group; each Rlais independently a Ci-io alkylene group, optionally substituted with one or more Xlagroups; each Xlagroup is independently selected from the group consisting of a Ci- io alkyl group, -C(O)ORlb, and -N(Rlc)(Rld), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)ORleand -N(Rlf)(R19); and

[0029] Rlb, Rlc, Rld, Rle, Rlfand R19, are each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group, the method comprising the steps of:

[0030] (a) providing a protein comprising one or more groups of Formula II, wherein:

[0031] X1and L1are as defined above, and reacting the protein comprising one or more groups of Formula II with a compound of Formula III, wherein :

[0032] Rlais as defined above;

[0033] R3ais selected from the group consisting of hydrogen and -SR3b; R3bis selected from the group consisting of hydrogen and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more X3agroups; each X3agroup is independently selected from the group consisting of a Ci- 10 alkyl group, -C(O)OR3c, and -N(R3d)(R3e), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)OR3fand -N(R3g)(R3h); and

[0034] R3c, R3d, R3e, R3f, R39and R3hare each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group, to provide a protein comprising one or more groups of Formula IV-A, wherein:

[0035] X1, L1and Rlaare as defined above, or a protein comprising one or more groups of Formula IV-B, wherein:

[0036] X1, L1, Rlaand R3bare as defined above;

[0037] (b) optionally, wherein when step (a) provides a protein comprising one or more groups of Formula IV-B, providing a protein comprising one or more groups of Formula IV- A by reducing the S-S bond in the one or more groups of Formula IV-B to provide a -SH group; and

[0038] (c) providing the modified protein comprising one or more groups of Formula I by nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A.

[0039] In a second aspect of the invention, there is provided a method of producing a modified protein, the modified protein comprising one or more groups of Formula V wherein: X5represents the peptide chain of the modified protein; each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group, which Ci-io alkylene group is optionally substituted with one or more X5agroups; each X5agroup is independently selected from the group consisting of - N(R5a)(R5b), and -OR5c;

[0040] R5a, R5b, and R5care each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f; and

[0041] R5d, R5e, and R5fare each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, the method comprising the steps of:

[0042] (d) providing a protein comprising one or more groups of Formula VI, x5- L5- NH2 VIwherein:

[0043] X5and L5are as defined above, and reacting the protein comprising one or more groups of Formula VI with 2- iminothiolane (Traut's reagent) to provide a protein comprising one or more groups of Formula VII, wherein:

[0044] X5and L5are as defined above; and

[0045] (e) providing the modified protein comprising one or more groups of Formula V by nitrosating the -SH group of the protein comprising one or more groups of Formula VII.

[0046] In a third aspect of the invention, there is provided a modified protein obtainable by a method according to the first or second aspects.

[0047] In a fourth aspect of the invention, there is provided a modified protein, wherein the modified protein comprises one or more groups of Formula I: wherein:

[0048] X1represents the peptide chain of the modified protein; each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group; each Rlais independently a Ci-io alkylene group, optionally substituted with one or more Xlagroups; each Xlagroup is independently selected from the group consisting of a Ci- 10 alkyl group, -C(O)ORlb, and -N(Rlc)(Rld), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)ORleand -N(Rlf)(R19); and

[0049] Rlb, Rlc, Rld, Rle, Rlfand R19, are each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group.

[0050] In a fifth aspect of the invention, there is provided a modified protein, wherein the modified protein comprises one or more groups of Formula V: wherein:

[0051] X5represents the peptide chain of the modified protein; each L5is a linker independently selected from the group consisting of a chemical bond, and a C1-10 alkylene group, which C1-10 alkylene group is optionally substituted with one or more X5agroups; each X5agroup is independently selected from the group consisting of - N(R5a)(R5b), and -OR5c;

[0052] R5a, R5b, and R5care each independently selected from the group consisting of hydrogen, and a C1-10 alkyl group, which C1-10 alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f; and

[0053] R5d, R5e, and R5fare each independently selected from the group consisting of hydrogen, and a C1-10 alkyl group. In a sixth aspect of the invention, there is provided a composition comprising the modified protein according to any one of the third to fifth aspects.

[0054] In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising the modified protein or the composition according to any one of the third to sixth aspects, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0055] In an eight aspect of the invention, there is provided a modified protein, composition, or pharmaceutical composition according to any one of the third to seventh aspects, which releases nitric oxide at physiological temperature.

[0056] In a ninth aspect of the invention, there is provided a modified protein, composition, or pharmaceutical composition according to any one of the third to eighth aspects, for use as a medicament.

[0057] In a tenth aspect of the invention, there is provided the modified protein, the composition, or the pharmaceutical composition according to any one of the third to eighth aspects, for use in wound healing, for use in tissue healing or for use in immune modulation.

[0058] In an eleventh aspect of the invention, there is provided the non-therapeutic use of the modified protein, composition, or pharmaceutical composition according to any one of the third to eighth aspects, in a cosmetic product.

[0059] DESCRIPTION OF THE FIGURES

[0060] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures in which:

[0061] Figure 1 shows the chromatograms from Fast Protein Liquid Chromatography (FPLC) analysis. (A) Presents the chromatogram from FPLC performed on filtered collagen. (B) Presents the chromatogram from FPLC analysis performed on a modified collagen sample which was synthesised as detailed in Example 2.

[0062] Figure 2 shows the absorption spectra (between 300 nm to 380 nm) of four samples of modified collagen and a reference sample of NaNOz. Each sample of modified collagen was synthesised as detailed in Example 2.

[0063] Figure 3 shows a chemiluminescence trace of NO signals detected from four samples of modified collagen, and from two control samples of NaNOz and S-Nitroso- / V- acetylpenicillamine (SNAP), stimulated by CuCl2 in the reaction chamber. The NO concentration generated from each sample of modified collagen is shown below each peak. Each modified collagen sample was synthesised as detailed in Example 2 and either stored as a liquid solution before analysis (Sample IDs: Jell 02, Jell 03 and Jell 04) or freeze- dried immediately after synthesis before being dissolved prior to analysis (Sample ID: 06501).

[0064] Figure 4 shows the results from nitrite assays utilising the Griess test for estimation of nitrite concentration. (A) Shows the calibration curve (absorbance vs nitrite concentration) generated from the nitrite assay of sodium nitrite standard solutions. (B) Shows the results from a nitrite assay performed on four samples of modified collagen. The sample IDs NN 5601, NN 5701 and NN S802 were each synthesised as detailed in Example 2. The sample ID NN 03801 was also synthesised as detailed in Example 2, however the activating agent used was EDC / NHS (instead of DMTMM). The chart shows the absorbance at 546 nm after performing the nitrite assay on each sample after it was left standing for 26 days at room temperature relative to the absorption recorded at day 0.

[0065] Figure 5 presents a plot of absorption at 546 nm against time in storage after performing a nitrite assay on four samples of modified collagen. The sample IDs NN5601 and NN5602 were stored in the presence of EDTA (10 mM) and at 5°C and -20°C respectively. The sample ID NN5603 was stored at 5°C and in the presence of NaCI (50 mM). The sample ID NN5604 was stored at 5°C and with no additives added. The four modified collagen samples were synthesised as detailed in Example 2.

[0066] Figure 6 shows the absorption at 546 nm after performing a nitrite assay on three freeze- dried samples of modified collagen. Each sample was stored at either 5°C, 20°C, or 37°C for 24 hours after synthesis. The modified collagen samples were synthesised as detailed in Example 2.

[0067] Figure 7 shows a myography trace of the vasodilatory effect of a sample of modified collagen in a porcine radial artery pre-contracted with phenylephrine (PE). The effect was completely reversed with the soluble guanylate cyclase (sGC) inhibitor, ODQ. sGC is the downstream enzyme that is stimulated by NO; reversal by this inhibitor confirms that the vasodilator effect is S-NO mediated. The modified collagen sample utilised was synthesised as detailed in Example 2.

[0068] Figure 8 shows a plot of log(A33s) vs. time for comparative samples of modified bovine collagen stored at room temperature or over ice. The comparative sample of modified bovine collagen was synthesised following the procedure described in WO 2016 / 166524. The absorbance at 335 nm (A335) corresponds to the characteristic UV absorbance of the S-NO bond in the comparative modified bovine collagen sample.

[0069] DETAILED DESCRIPTION OF THE INVENTION

[0070] In a first aspect of the invention, there is provided a method of producing a modified protein, the modified protein comprising one or more groups of Formula I

[0071] I wherein:

[0072] X1represents the peptide chain of the modified protein; each L1is a linker independently selected from the group consisting of a chemical bond, and a C1-10 alkylene group; each Rlais independently a C1-10 alkylene group, optionally substituted with one or more Xlagroups; each Xlagroup is independently selected from the group consisting of a Ci-

[0073] 10 alkyl group, -C(O)ORlb, and -N(Rlc)(Rld), which C1-10 alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)ORleand -N(Rlf)(R19); and

[0074] Rlb, Rlc, Rld, Rle, Rlfand R19, are each independently selected from the group consisting of hydrogen, a C1-10 alkyl group, and a C3-6 cycloalkyl group, the method comprising the steps of:

[0075] (a) providing a protein comprising one or more groups of Formula II, wherein:

[0076] X1and L1are as defined above, and reacting the protein comprising one or more groups of Formula II with a compound of Formula III, wherein :

[0077] Rlais as defined above;

[0078] R3ais selected from the group consisting of hydrogen and -SR3b;

[0079] R3bis selected from the group consisting of hydrogen and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more X3agroups; each X3agroup is independently selected from the group consisting of a Ci- 10 alkyl group, -C(O)OR3c, and -N(R3d)(R3e), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)OR3fand -N(R3g)(R3b); and

[0080] R3c, R3d, R3e, R3f, R39and R3bare each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group, to provide a protein comprising one or more groups of Formula IV-A, wherein:

[0081] X1, L1and Rlaare as defined above, or a protein comprising one or more groups of Formula IV-B, wherein:

[0082] X1, L1, Rlaand R3bare as defined above;

[0083] (b) optionally, wherein when step (a) provides a protein comprising one or more groups of Formula IV-B, providing a protein comprising one or more groups of Formula IV- A by reducing the S-S bond in the one or more groups of Formula IV-B to provide a -SH group; and

[0084] (c) providing the modified protein comprising one or more groups of Formula I by nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A.

[0085] When used herein, "peptide chain" refers to a linear arrangement of amino acids within the protein in which the amino acids are linked together by peptide bonds. It will be understood that each X1in the Formulae above independently represent a carbon within in the peptide chain of the protein upon which one of the groups of Formula I to IV is attached. Each X1independently represents an alpha-carbon of an amino-acid within the protein's peptide chain and is not a carbon atom in a side chain of an amino acid.

[0086] Amino acids are organic compounds that contain amino (-NH2) and carboxyl (-CO2H) functional groups, along with (in most cases) a sidechain. Alpha amino acids are those where the amino and carboxyl functional groups are bonded to the same carbon atom (i.e. the alpha-carbon atom). Particular alpha amino acids that might be mentioned are the proteinogenic amino acids. Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during translation. The proteinogenic amino acids include glycine (Gly), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), pyrrolysine (Pyl), selenocysteine (Sec), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). Unless otherwise specified, alkyl groups defined herein may be straight-chain or, when there is a sufficient number of carbon atoms, be branched-chain.

[0087] When used herein, "alkylene" (i.e. alkanediyl) refers to a divalent alkyl group. Particular alkylene groups that may be mentioned include, for example, propylene (n-propylene or isopropylene), ethylene and, particularly, methylene (i.e. -CH2-).

[0088] When there is a sufficient number (i.e. a minimum of three, as appropriate) of carbon atoms, an alkyl group may be cyclic, so forming a cycloalkyl group. Cycloalkyl groups that may be mentioned include monocyclic groups, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Such cycloalkyl groups may be bridged (so forming, for example, fused ring systems such as three fused cycloalkyl groups). Further, such cycloalkyl groups may be saturated or unsaturated containing, e.g., one or more double bonds (forming for example a cycloalkenyl). Further, where there is a sufficient number (i.e. a minimum of four) such cycloalkyl groups may also be part cyclic, e.g. forming an alkylene-cycloalkyl group (for example, -CH2-C3H5). The points of attachment of cycloalkyl groups may be via any atom in the ring system. For the avoidance of doubt, optional substituents may also be other cyclic groups, which may be attached via a single carbon atom common to both rings, so forming a spiro-cycle.

[0089] For the avoidance of doubt, in cases in which the identity of two or more substituents may be the same, the actual identities of the respective substituents are not in any way interdependent.

[0090] Where groups are referred to herein as being optionally substituted it is specifically contemplated that such optional substituents may be not present (i.e. references to such optional substituents may be removed), in which case the optionally substituted group may be referred to as being unsubstituted in certain embodiments.

[0091] Throughout this specification, structures may or may not be presented with chemical names. Where any question arises as to nomenclature, the structure prevails.

[0092] Unless indicated otherwise, all technical and scientific terms used herein will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0093] For the avoidance of doubt, it will be understood that references herein to particular aspects of the invention (such as the first aspect of the invention) will include references to all embodiments and particular features thereof, which embodiments and particular features may be taken in combination to form further embodiments and features of the invention.

[0094] In preferred embodiments of the first aspect, each X1independently represents an alphacarbon of an amino-acid within the protein's peptide chain.

[0095] In some embodiments of the first aspect, the peptide chain represented by X1is a peptide chain consisting of only proteinogenic amino acids. In other embodiments of the first aspect, the peptide chain represented by X1is a peptide chain comprising both proteinogenic and non-proteinogenic amino acids. In some embodiments of the first aspect, the peptide chain represented by X1is from natural or recombinant sources.

[0096] In certain embodiments of the first aspect, the peptide chain represented by X1is from a mammalian source. In some embodiments, the mammalian source is cow, horse, pig, human, rat, sheep, or goat.

[0097] In some embodiments of the first aspect, the peptide chain represented by X1is from a non-mammalian source. In some embodiments, the non-mammalian source is jellyfish, marine invertebrate, fish, or amphibians.

[0098] In preferred embodiments of the first aspect, the peptide chain represented by X1is from a non-mammalian source, wherein the non-mammalian source is jellyfish. In some embodiments, the jellyfish is from the sub-phylum Scyphozoa.

[0099] In certain embodiments of the first aspect, the peptide chain represented by X1is from the jellyfish sub-phylum Scyphozoa, wherein the Scyphozoa is selected from the group consisting of: the order Rhizostomeae, including, but not limited to, Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Cassiopea sp. (upside-down jellyfish), including but not limited to Cassiopea andromeda, the order Semaeostomease, including Aurelia sp., and other species such as Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, or any combination thereof. Preferably, the source of the jellyfish collagen is Rhizostomas pulmo.

[0100] In some embodiments of the first aspect, the peptide chain represented by X1is selected from the group of proteins consisting of collagen, fibronectin, keratin, silk, resilin, laminin, and elastin.

[0101] In preferred embodiments of the first aspect, the peptide chain represented by X1is collagen. In some embodiments, the collagen is any one of the following: collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI, or XVII.

[0102] It will be understood that in embodiments of the first aspect wherein the peptide chain represented by X1is collagen, the method of producing a modified protein according to the first aspect is a method of producing a modified collagen, wherein the modified collagen comprises one or more groups of Formula I.

[0103] In some embodiments of the first aspect, each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-6 alkylene group, preferably each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, more preferably each L1is a linker independently selected from the group consisting of a chemical bond, methylene, and ethylene, most preferably each L1is a linker independently selected from methylene, and ethylene.

[0104] In some embodiments of the first aspect, the protein comprising one or more groups of Formula II is a protein comprising one or more groups selected from the Formulae consisting of Formula II-A, Formula II-B, and Formula II-C, wherein:

[0105] X1is as defined above.

[0106] In certain embodiments of the first aspect, the protein comprising one or more groups of Formula II is a protein comprising one or more groups selected from the Formulae consisting of Formula II-B, and Formula II-C.

[0107] In some embodiments of the first aspect, R3ais -SR3b.

[0108] In some embodiments of the first aspect, R3ais hydrogen.

[0109] In some embodiments of the first aspect, R3bis a Ci-io alky group, optionally substituted with one or more X3agroups.

[0110] In preferred embodiments of the first aspect, R3bis a Ci-6 alkyl group, for example a Ci-4 alkyl group, such as a methyl group or an ethyl group, each optionally substituted with one or more X3agroups.

[0111] In some embodiments of the first aspect, each X3agroup is independently selected from the group consisting of a Ci-io alkyl group, -C(O)OH and -NH2.

[0112] In some embodiments of the first aspect, each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0113] In some embodiments of the first aspect, R3bis a C1-10 alkyl group, optionally substituted with one or more X3agroups, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0114] In certain embodiments of the first aspect, R3bis a C1-6 alkyl group, for example a Ci-4 alkyl group, such as a methyl group or an ethyl group, each optionally substituted with one or more X3agroups, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0115] In some embodiments of the first aspect, each Rlais a C1-6 alkylene group, for example a Ci-4 alkylene group, such as a methylene group or an ethylene group, each optionally substituted with one or more Xlagroups.

[0116] In some embodiments of the first aspect, each Xlagroup is independently selected from the group consisting of a C1-10 alkyl group, -C(O)OH and -NH2.

[0117] In preferred embodiments of the first aspect, each Xlagroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0118] In more preferred embodiments of the first aspect, each Xlagroup is -C(O)OH. In some embodiments of the first aspect, each Rlais a Ci-io alkylene group optionally substituted with one or more -C(O)OH groups.

[0119] In preferred embodiments of the first aspect, each Rlais a Ci-6 alkylene group, for example a Ci-4 alkylene group, such as a methylene group or an ethylene group, each optionally substituted with one or more -C(O)OH groups.

[0120] In certain embodiments of the first aspect, each Rlais a Ci-io alkylene group optionally substituted with one or more -C(O)OH groups, and R3bis a Ci-io alkyl group, optionally substituted with one or more X3agroups, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0121] In preferred embodiments of the first aspect, each Rlais a C1-6 alkylene group optionally substituted with one or more -C(O)OH groups, and R3bis a C1-6 alkyl group, optionally substituted with one or more X3agroups, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0122] In more preferred embodiments of the first aspect, each Rlais a Ci-4 alkylene group optionally substituted with one or more -C(O)OH groups, and R3bis a Ci-4 alkyl group, optionally substituted with one or more X3agroups, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0123] In yet more preferred embodiments of the first aspect, each Rlais a methylene group or an ethylene group, optionally substituted with one or more -C(O)OH groups, and R3bis a methyl group or an ethyl group, optionally substituted with one or more X3agroups, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0124] In some embodiments of the first aspect, the compound of Formula III is a compound selected from the group consisting of:

[0125] In certain embodiments of the first aspect, the compound of Formula III is the compound, In some embodiments of the first aspect, the compound of Formula III is a compound selected from the group consisting of: nd

[0126] In certain embodiments of the first aspect, the compound of Formula III is the compound,

[0127] In some embodiments of the first aspect, step (a) is performed in the presence of an activating agent.

[0128] In certain embodiments of the first aspect, wherein step (a) is performed in the presence of an activating agent, the activating agent is selected from the group consisting of 4-(4,6- dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), 4- dimethylaminopyridine (DMAP), and a combination of l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0129] In preferred embodiments of the first aspect, wherein step (a) is performed in the presence of an activating agent, the activating agent is selected from the group consisting of DMTMM and a combination of EDC and NHS.

[0130] When the activating agent used is a combination of EDC and NHS, it is believed that the carboxyl group(s) in the one or more groups of Formula II reacts with the combination of EDC and NHS to form a O-acyl-isourea intermediate (NHS), which then spontaneously reacts with the amine group in the compound of Formula III to form an amide bond and an isourea by-product.

[0131] When the activating agent used is DMTMM, it is believed that the carboxyl group(s) in the one or more groups of Formula II reacts with DMTMM to form an active ester, releasing a molecule of N-methylmorpholinium (NMM). The resulting activated ester is highly reactive and rapidly undergoes a nucleophilic attack by the amine group in the compound of Formula III to form an amide link and a soluble by-product, a molecule of 4,6-dimethoxy- l,3,5-triazin-2-ol. In preferred embodiments of the first aspect, step (a) is performed in the presence of the activating agent DMTMM.

[0132] In some embodiments of the first aspect, step (a) is performed at about pH 7.0 or less, at about pH 6.5 or less, at about pH 6.0 or less, at about pH 5.5 or less, at about pH 5.0 or less, at about pH 4.5 or less, at about pH 4.0 or less, at about pH 3.5 or less, at about pH 3.0 or less, at about pH 2.5 or less, at about pH 2.0 or less, at about pH 1.5 or less, at about pH 1.0 or less, or at about pH 0.5 or less. Preferably, step (a) is performed at about pH 7.0 or less, more preferably at about pH 6.5 or less, most preferably at about pH 6.0 or less.

[0133] In other embodiments of the first aspect, step (a) is performed at from about pH 0.5 to about 7.0, at from about pH 1.0 to about 6.5, at from about pH 1.5 to about 6.0, at from about pH 2.0 to about 5.5, at from about pH 2.5 to about 5.0, at from about pH 3.0 to about 4.5, at from about pH 3.5 to about 4.0, or at from about pH 3.0 to about 7.0, at from about pH 3.5 to about 7.0, at from about pH 4.0 to about 7.0, at from about pH 4.5 to about 7.0, at from about pH 5.0 to about 7.0, at from about pH 5.5 to about 7.0, at from about pH 6.0 to about 7.0, or at from about pH 6.5 to about 7.0. Preferably, step (a) is performed at from about pH 1.0 to about 7.0, more preferably at from about pH 3.0 to about 7.0, yet more preferably at from about pH 4.0 to about 6.0, most preferably around at about pH 5.0.

[0134] In some embodiments of the first aspect, the protein comprising one or more groups of Formula IV-A, or the protein comprising one or more groups of Formula IV-B, obtained from step (a) is purified before either step (b) or step (c) is performed.

[0135] In certain embodiments of the first aspect, the protein comprising one or more groups of Formula IV-A, or the protein comprising one or more groups of Formula IV-B, obtained from step (a) is purified by dialysis before either step (b) or step (c) is performed. Preferably the dialysis is carried out using a solution comprising acetic acid and sodium chloride.

[0136] In some embodiments of the first aspect, step (b) comprises reacting a protein comprising one or more groups of Formula IV-B with a reducing agent.

[0137] In certain embodiments of the first aspect, wherein step (b) comprises reacting a protein comprising one or more groups of Formula IV-B with a reducing agent, the reducing agent is selected from the group consisting of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT), and (2S)-2-amino-l,4-dimercaptobutane (DTBA).

[0138] In preferred embodiments of the first aspect, wherein step (b) comprises reacting a protein comprising one or more groups of Formula IV-B with a reducing agent, the reducing agent is selected from the group consisting of DTT, and TCEP.

[0139] In more preferred embodiments of the first aspect, step (b) comprises reacting a protein comprising one or more groups of Formula IV-B with the reducing agent is TCEP.

[0140] In some embodiments of the first aspect, step (b) comprises reacting a protein comprising one or more groups of Formula IV-B with the reducing agent TCEP under vacuum and at approximately 5°C.

[0141] In some embodiments of the first aspect, step (b) is performed at about pH 7.0 or less, at about pH 6.5 or less, at about pH 6.0 or less, at about pH 5.5 or less, at about pH 5.0 or less, at about pH 4.5 or less, at about pH 4.0 or less, at about pH 3.5 or less, at about pH 3.0 or less, at about pH 2.5 or less, at about pH 2.0 or less, at about pH 1.5 or less, at about pH 1.0 or less, or at about pH 0.5 or less. Preferably, step (b) is performed at about pH 5.0 or less, more preferably at about pH 4.5 or less, most preferably at about pH 4.0 or less.

[0142] In other embodiments of the first aspect, step (b) is performed at from about pH 0.5 to about 7.0, at from about pH 1.0 to about 6.5, at from about pH 1.5 to about 6.0, at from about pH 2.0 to about 5.5, at from about pH 2.5 to about 5.0, at from about pH 3.0 to about 4.5, at from about pH 3.5 to about 4.0, or at from about pH 1.0 to about 7.0, at from about pH 1.5 to about 7.0, at from about pH 2.0 to about 7.0, at from about pH 2.5 to about 7.0, at from about pH 3.0 to about pH 7.0, at from about pH 3.5 to about 7.0, at from about pH 4.0 to about 7.0, at from about pH 4.5 to about 7.0, at from about pH 5.0 to about 7.0, at from about pH 5.5 to about 7.0, at from about pH 6.0 to about 7.0, or at from about pH 6.5 to about 7.0. Preferably, step (b) is performed at from about pH 1.0 to about 7.0, more preferably at from about pH 1.5 to about 7.0, yet more preferably at from about pH 2.0 to about 5.5, most preferably at around pH 3.0.

[0143] In some embodiments of the first aspect, step (c) is carried out on the unpurified product obtained from step (b).

[0144] In other embodiments of the first aspect, the protein comprising one or more groups of Formula IV-A obtained from step (b) is purified (for example, by dialysis) before step (c) is performed.

[0145] In some embodiments of the first aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A in step (c) can be carried out by any technique known in the art for S-nitrosation to yield a S-nitroso group.

[0146] When used herein "S-nitroso", "S-nitrosothiol" and "thionitrites" groups all refer to the same functional group containing a nitroso group attached to the sulphur atom of a thiol and having general formula § N=OEac|nterm maype usec| interchangeably.

[0147] In preferred embodiments of the first aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A in step (c) is carried out at below room temperature, such as a temperature of approximately 5°C.

[0148] In some embodiments of the first aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A in step (c) comprises reacting the protein with one or more reagents selected from the group of reagents consisting of a solution of acidified nitrite, gaseous NO, and a S-nitrosothiol.

[0149] In preferred embodiments of the first aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A in step (c) comprises reacting the protein with a solution of acidified nitrite, for example acidified sodium or potassium nitrite, preferably acidified sodium nitrite.

[0150] Step (c) can be monitored spectrophotometrically in the presence and absence of copper ions, which are known to selectively release NO from S-nitrosothiol groups. Typically, S- NO bonds lead to specific absorption peaks in the range of 330-340 nm and in the range of 540-600 nm in the UV-visible spectrum. For example, a modified collagen comprising S-nitroso groups produced in accordance with WO 2016 / 16166524 displayed an absorption peak at around 335 nm. In some embodiments of the first aspect, the modified protein comprising one or more groups of Formula I provided by step (c) is purified.

[0151] In certain embodiments of the first aspect, the modified protein comprising one or more groups of Formula I provided by step (c) is purified by dialysis, preferably the dialysis is carried out using a solution comprising acetic acid and sodium chloride.

[0152] In a second aspect of the invention, there is provided a method of producing a modified protein, the modified protein comprising one or more groups of Formula V wherein:

[0153] X5represents the peptide chain of the modified protein; each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group, which Ci-io alkylene group is optionally substituted with one or more X5agroups; each X5agroup is independently selected from the group consisting of - N(R5a)(R5b), and -OR5c;

[0154] R5a, R5b, and R5care each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f; and

[0155] R5d, R5e, and R5fare each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, the method comprising the steps of:

[0156] (d) providing a protein comprising one or more groups of Formula VI,

[0157] X5- L5- NH2 wherein:

[0158] X5and L5are as defined above, and reacting the protein comprising one or more groups of Formula VI with 2-iminothiolane (Traut's reagent) to provide a protein comprising one or more groups of Formula VII, wherein:

[0159] X5and L5are as defined above; and

[0160] (e) providing the modified protein comprising one or more groups of Formula V by nitrosating the -SH group of the protein comprising one or more groups of Formula VII.

[0161] When used herein, "peptide chain" refers to a linear arrangement of amino acids within the protein in which the amino acids are linked together by peptide bonds. Therefore, it will be understood that each X5in the Formulae above independently represent a carbon within in the peptide chain of the protein upon which one of the groups of Formula V to VII is attached. Each X5independently represents an alpha-carbon of an amino-acid within the protein's peptide chain and is not a carbon atom in a side chain of an amino acid.

[0162] In preferred embodiments of the second aspect, each X5independently represents an alpha-carbon of an amino-acid within the protein's peptide chain.

[0163] In some embodiments of the second aspect, the peptide chain represented by X5is a peptide chain consisting of only proteinogenic amino acids. In other embodiments of the second aspect, the peptide chain represented by X5is a peptide chain comprising both proteinogenic and non-proteinogenic amino acids.

[0164] In some embodiments of the second aspect, the peptide chain represented by X5is from natural or recombinant sources.

[0165] In certain embodiments of the second aspect, the peptide chain represented by X5is from a mammalian source. In some embodiments, the mammalian source is cow, horse, pig, human, rat, sheep, or goat.

[0166] In some embodiments of the second aspect, the peptide chain represented by X5is from a non-mammalian source. In some embodiments, the non-mammalian source is jellyfish, marine invertebrate, fish, or amphibians.

[0167] In preferred embodiments of the second aspect, the peptide chain represented by X5is from a non-mammalian source, wherein the non-mammalian source is jellyfish. In some embodiments, the jellyfish is from the sub-phylum Scyphozoa.

[0168] In certain embodiments of the second aspect, the peptide chain represented by X5is from the jellyfish sub-phylum Scyphozoa, wherein the Scyphozoa is selected from the group consisting of: the order Rhizostomeae, including, but not limited to, Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Cassiopea sp. (upside-down jellyfish), including but not limited to Cassiopea andromeda, the order Semaeostomease, including Aurelia sp., and other species such as Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, or any combination thereof. Preferably the source of the jellyfish collagen is Rhizostomas pulmo.

[0169] In some embodiments of the second aspect, the peptide chain represented by X5is selected from the group of proteins consisting of collagen, fibronectin, keratin, silk, resilin, laminin, and elastin.

[0170] In preferred embodiments of the second aspect, the peptide chain represented by X5is collagen. In some embodiments, the collagen is any one of the following: collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI, or XVII.

[0171] It will be understood that in embodiments of the second aspect wherein the peptide chain represented by X5is collagen, the method of producing a modified protein according to the second aspect is a method of producing a modified collagen, wherein the modified collagen comprises one or more groups of Formula V.

[0172] In some embodiments of the second aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-6 alkylene group, which Ci-6 alkylene group is optionally substituted with one or more X5agroups.

[0173] In preferred embodiments of the second aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, which Ci-4 alkylene group is optionally substituted with one or more X5agroups.

[0174] In some embodiments of the second aspect, each X5agroup is independently selected from the group consisting of -NH(R5b), and -OH, wherein each R5bis independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f.

[0175] In preferred embodiment of the second aspect, each R5bis independently a Ci-6 alkyl group, for example a Ci-4 alkyl group, such as a methyl group or an ethyl group, each optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f.

[0176] In more preferred embodiments of the second aspect, each R5bis a methyl group optionally substituted with one or more groups selected from the group consisting of -NH2, and =NH.

[0177] In certain embodiments of the second aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, which Ci-4 alkylene group is optionally substituted with one or more X5agroups, wherein each X5agroup is independently selected from the group consisting of -NH(R5b), and -OH, wherein each R5bis a methylene group optionally substituted with one or more groups selected from the group consisting of -NH2, and =NH.

[0178] In preferred embodiments of the second aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, which Ci-4 alkylene group is optionally substituted with one or more -OH groups.

[0179] In some embodiments of the second aspect, the protein comprising one or more groups of Formula VI is a protein comprising one or more groups selected from the Formulae consisting of Formula VI-A, Formula VI-B, Formula VI-C, and Formula VI-D: - wherein:

[0180] X5is as defined above.

[0181] In preferred embodiments of the second aspect, the protein comprising one or more groups of Formula VI is a protein comprising one or more groups selected from the Formulae consisting of Formula VI-A, Formula VI-B, and Formula VI-C.

[0182] Traut's reagent, 2-iminothiolane, is a small thiolation compound that reacts spontaneously and efficiently with primary amines in a ring-opening reaction to introduce terminal thiol (-SH) groups. It's synthesis and application were first disclosed by Traut R R, Bollen A, Sun T, et al. in a 1973 paper titled 'Methyl 4-mercaptobutyrimidate as a cleavable crosslinking reagent and its application to the Escherichia coli 30S ribosome' in Biochemistry 12(17):3266-3273. Today, Traut's reagent is readily available commercially, (e.g., from Thermo Fisher Scientific)

[0183] The chemical structure of Traut's reagent and its general reaction scheme with molecules containing primary amines is shown in Scheme 1 below:

[0184] Traut's ReagentPnw®neModification producing a terminal thiol group

[0185] Scheme 1 - Structure of Traut's Reagent and reaction scheme with primary amines. The amount of Traut's reagent required in step (d) depends upon the protein size, concentration, and level of desired thiolation.

[0186] In some embodiments of the second aspect, the molar excess of Traut's reagent used in step (d) is about 1.0 or more, is about 1.5 or more, is about 2.0 or more, is about 2.5 or more, is about 3.0 or more, is about 3.5 or more, is about 4.0 or more, is about 4.5 or more, is about 5.0 or more, is about 6.0 or more, is about 7.0 or more, is about 8.0 or more, is about 9.0 or more, is about 10.0 or more, is about 12.0 or more, is about 14.0 or more, is about 16.0 or more, is about 18.0 or more, or is about 20.0 or more molar excess. Preferably, the molar excess of Traut's reagent used in step (d) is about 1.0 or more, more more preferably is about 1.5 or more, most preferably is about 2.0 or more.

[0187] In some embodiments of the second aspect, the molar excess of Traut's reagent used in step (d) is from about 1.0 to about 100, is from about 1.5 to about 90, is from about 2.0 to about 80, is from about 2.5 to about 70, is from about 3.0 to about 60, is from about 3.5 to about 50, is from about 4.0 to about 40, is from about 4.5 to about 30, is from about 5.0 to about 20 or is from about 2.0 to about 100, is from about 3.0 to about 100, is from about 4.0 to about 100, is from about 5.0 to about 100, is from about 6.0 to about 100, is from about 7.0 to about 100, is from about 8.0 to about 100, is from about 9.0 to about 100, is from about 10.0 to about 100, is from about 15 to about 100, is from about 20 to about 100, is from about 30 to about 100, is from about 40 to about 100, is from about 50 to about 100, is from about 70 to about 100, is from about 90 to about 100. Preferably the molar excess of Traut's reagent used in step (d) is from about 1.0 to about 100, more preferably is from about 2.0 to about 100, yet more preferably is from about 2.0 to about 80, most preferably is from about 2.0 to about 20.

[0188] In some embodiments of the second aspect, step (d) performed at about pH 9.0 or less, at about pH 8.5 or less, at about pH 8.0 or less, at about pH 7.5 or less, at about pH 7.0 or less, at about pH 6.5 or less, at about pH 6.0 or less, at about pH 5.5 or less, at about pH 5.0 or less, at about pH 4.5 or less, at about pH 4.0 or less, at about pH 3.5 or less, at about pH 3.0 or less. Preferably, step (d) is performed at about pH 9.0 or less, more preferably at about pH 8.5 or less, most preferably at about pH 8.0.

[0189] In other embodiments of the second aspect, step (d) is performed at from about pH 3.0 to about 9.0, at from about pH 3.5 to about 8.5, at from about pH 4.0 to about 8.0, at from about pH 4.5 to about 7.5, at from about pH 5.0 to about 7.0, at from about pH 5.5 to about 6.5, or at from about pH 3.5 to about 9.0, at from about pH 4.0 to about 9.0, at from about pH 4.5 to about 9.0, at from about pH 5.0 to about 9.0, at from about pH 5.5 to about 9.0, at from about pH 6.0 to about 9.0, at from about pH 6.5 to about 9.0, at from about pH 7.0 to about 9.0, at from about pH 7.5 to about 9.0, at from about pH 8.0 to about 9.0. Preferably, step (d) is performed at from about pH 3.0 to about 9.0, more preferably at from about pH 4.0 to about 9.0, yet more preferably at from about pH 5.0 to about 9.0, most preferably at about pH 8.0.

[0190] In some embodiments of the second aspect, the protein comprising one or more groups of Formula VII obtained from step (d) is purified before step (e) is performed.

[0191] In other embodiments of the second aspect, step (e) is performed on the unpurified product of step (d).

[0192] In preferred embodiments of the second aspect, the protein comprising one or more groups of Formula VII obtained from step (d) is purified by dialysis before step (e) is performed. Preferably the dialysis is carried out using a solution comprising acetic acid and sodium chloride.

[0193] In some embodiments of the second aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula VII in step (e) can be carried out by any technique known in the art for S-nitrosation to yield a S-nitroso group.

[0194] In preferred embodiments of the second aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula VII in step (e) is carried out at below room temperature, such as a temperature of approximately 5°C.

[0195] In some embodiments of the second aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula VII in step (e) comprises reacting the protein with one or more reagents selected from the group of reagents consisting of a solution of acidified nitrite, gaseous NO, and a S-nitrosothiol.

[0196] In preferred embodiments of the second aspect, nitrosating the -SH group of the protein comprising one or more groups of Formula VII in step (e) comprises reacting the protein with a solution of acidified nitrite, for example acidified sodium or potassium nitrite, preferably acidified sodium nitrite.

[0197] Step (e) can be monitored spectrophotometrically in the presence and absence of copper ions, which are known to selectively release NO from S-nitrosothiol groups. Typically, S- NO bonds lead to specific absorption peaks in the range of 330-340nm and in the range of 540-600 nm in the UV-visible spectrum. For instance, a modified collagen comprising S-nitroso groups manufactured in WO 2016 / 16166524 displayed an absorption peak at around 335 nm.

[0198] In some embodiments of the second aspect, the modified protein comprising one or more groups of Formula V provided by step (e) is purified.

[0199] In certain embodiments of the second aspect, the modified protein comprising one or more groups of Formula V provided by step (e) is purified by dialysis, preferably the dialysis is carried out using a solution comprising acetic acid and sodium chloride.

[0200] Further embodiments according to both the first and second aspects of the invention will now be described in further detail.

[0201] In some embodiments of the first and second aspects, the method further comprises the step of:

[0202] (i) Incorporating the modified protein into one or more of the groups comprising a collagen and a gelatin matrix; or

[0203] (ii) Incorporating the modified protein into a polymer.

[0204] In certain embodiments of the first and second aspects which further comprise step (i) as defined above, the collagen is derived from a jellyfish, such as Rhizostoma pulmo jellyfish tissue i.e. Barrel jellyfish.

[0205] In some embodiments of the first and second aspects, the modified protein can either be freeze-dried or kept as a liquid composition.

[0206] In preferred embodiments of the first and second aspects, the modified protein is freeze dried. As is shown in the Examples below, the stability of the S-nitroso groups in the modified proteins comprising one or more groups of Formula I, or the modified proteins comprising one or more groups of Formula V, are improved when stored as a freeze-dried sample relative to a liquid sample.

[0207] In some embodiments of the first and second aspects, the method of producing a modified protein is performed in an aqueous environment, preferably, the aqueous environment is substantially free of dimethyl sulfoxide (DMSO), even more preferably the aqueous environment contains no DMSO.

[0208] By "substantially free of" we include the meaning that there is less than 10 weight % of the stated component. Preferably there is less than 9, 8, 7, 6, 5, 4, 3 weight % of the stated component. More preferably there is less than 2 weight % of the stated component. Yet more preferably there is less than 1 weight % of the stated component.

[0209] In preferred embodiments of the first and second aspects, the peptide chain represented by X1or X5is soluble in water. It may be that in some embodiments the pH of the solution is modified to maintain the solubility of the peptide chain represented by X1or X5.

[0210] Surprisingly, the method of the first and second aspects of the invention allow for the effective production of modified proteins comprising S-nitrosothiol groups (for example a modified collagen comprising one or more groups of Formula I, or a modified collagen comprising one or more groups of Formula V) under aqueous conditions and with good yields, whilst also resulting in stable S-nitrosothiol groups that are capable of releasing nitric oxide after storage of the modified protein for a period of days, weeks and months.

[0211] According to a third aspect of the invention, there is provided a modified protein obtainable by a method according to the first or second aspect of the invention.

[0212] In a fourth aspect of the invention, there is provided a modified protein, wherein the modified protein comprises one or more groups of Formula I: wherein:

[0213] X1represents the peptide chain of the modified protein; each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group; each Rlais independently a Ci-io alkylene group, optionally substituted with one or more Xlagroups; each Xlagroup is independently selected from the group consisting of a Ci- io alkyl group, -C(O)ORlb, and -N(Rlc)(Rld), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)ORleand -N(Rlf)(R19); and Rlb, Rlc, Rld, Rle, Rlfand R19, are each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group.]

[0214] In preferred embodiments of the fourth aspect, each X1independently represents an alpha-carbon of an amino-acid within the modified protein's peptide chain.

[0215] In some embodiments of the fourth aspect, the peptide chain represented by X1is a peptide chain consisting of only proteinogenic amino acids. In other embodiments of the fourth aspect, the peptide chain represented by X1is a peptide chain comprising both proteinogenic and non-proteinogenic amino acids.

[0216] In some embodiments of the fourth aspect, the peptide chain represented by X1is from natural or recombinant sources.

[0217] In certain embodiments of the fourth aspect, the peptide chain represented by X1is from a mammalian source. In some embodiments, the mammalian source is cow, horse, pig, human, rat, sheep, or goat.

[0218] In some embodiments of the fourth aspect, the peptide chain represented by X1is from a non-mammalian source. In some embodiments, the non-mammalian source is jellyfish, marine invertebrate, fish, or amphibians.

[0219] In preferred embodiments of the fourth aspect, the peptide chain represented by X1is from a non-mammalian source, wherein the non-mammalian source is jellyfish. In some embodiments, the jellyfish is from the sub-phylum Scyphozoa.

[0220] In certain embodiments of the fourth aspect, the peptide chain represented by X1is from the jellyfish sub-phylum Scyphozoa, wherein the Scyphozoa is selected from the group consisting of: the order Rhizostomeae, including, but not limited to, Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Cassiopea sp. (upside-down jellyfish), including but not limited to Cassiopea andromeda, the order Semaeostomease, including Aurelia sp., and other species such as Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, or any combination thereof. Preferably the source of the jellyfish collagen is Rhizostomas pulmo.

[0221] In some embodiments of the fourth aspect, the peptide chain represented by X1is selected from the group of proteins consisting of collagen, fibronectin, keratin, silk, resilin, laminin, and elastin.

[0222] In preferred embodiments of the fourth aspect, the peptide chain represented by X1is collagen. In some embodiments, the collagen is any one of the following: collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI, or XVII.

[0223] It will be understood that in embodiments of the fourth aspect wherein the peptide chain represented by X1is collagen, the modified protein according to the fourth aspect is a modified collagen, wherein the modified collagen comprises one or more groups of Formula I.

[0224] In some embodiments of the fourth aspect, each L1is a linker independently selected from the group consisting of a chemical bond, and a C1-6 alkylene group, preferably each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, more preferably each L1is a linker independently selected from the group consisting of a chemical bond, methylene, and ethylene, most preferably each L1is a linker independently selected from methylene, and ethylene.

[0225] In some embodiments of the fourth aspect, the modified protein comprising one or more groups of Formula I is a modified protein comprising one or more groups selected from the group of Formulae consisting of Formulae I-A, Formula I-B, and Formula I-C: wherein:

[0226] X1, and Rlaare as defined above.

[0227] In certain embodiments of the fourth aspect, the modified protein comprising one or more groups of Formula I is a modified protein comprising one or more groups selected from the group of Formulae consisting of Formula I-B, and Formula I-C.

[0228] In some embodiments of the fourth aspect, each Rlais a Ci-6 alkylene group, for example a Ci-4 alkylene group, such as a methylene group or an ethylene group, each optionally substituted with one or more Xlagroups.

[0229] In some embodiments of the fourth aspect, each Xlagroup is independently selected from the group consisting of a Ci-io alkyl group, -C(O)OH and -NH2.

[0230] In preferred embodiments of the fourth aspect, each Xlagroup is independently selected from the group consisting of -C(O)OH and -NH2.

[0231] In more preferred embodiments of the fourth aspect, each Xlagroup is -C(O)OH.

[0232] In some embodiments of the fourth aspect, each Rlais a C1-10 alkylene group optionally substituted with one or more -C(O)OH groups.

[0233] In more preferred embodiments of the fourth aspect, each Rlais a C1-6 alkylene group, for example a C1-4 alkylene group, such as a methylene group or an ethylene group, each optionally substituted with one or more -C(O)OH groups.

[0234] In preferred embodiments of the fourth aspect, each Rlais ethylene, optionally substituted with a -C(O)OH group.

[0235] In a fifth aspect of the invention, there is provided a modified protein, wherein the modified protein comprises one or more groups of Formula V: wherein:

[0236] X5represents the peptide chain of the modified protein; each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group, which Ci-io alkylene group is optionally substituted with one or more X5agroups; each X5agroup is independently selected from the group consisting of - N(R5a)(R5b), and -OR5c;

[0237] R5a, R5b, and R5care each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f; and

[0238] R5d, R5e, and R5fare each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group.

[0239] In preferred embodiments of the fifth aspect, each X5independently represents an alphacarbon of an amino-acid within the modified protein's peptide chain.

[0240] In some embodiments of the fifth aspect, the peptide chain represented by X5is a peptide chain consisting of only proteinogenic amino acids. In other embodiments of the fifth aspect, the peptide chain represented by X5is a peptide chain comprising both proteinogenic and non-proteinogenic amino acids.

[0241] In some embodiments of the fifth aspect, the peptide chain represented by X5is from natural or recombinant sources.

[0242] In certain embodiments of the fifth aspect, the peptide chain represented by X5is from a mammalian source. In some embodiments, the mammalian source is cow, horse, pig, human, rat, sheep, or goat.

[0243] In some embodiments of the fifth aspect, the peptide chain represented by X5is from a non-mammalian source. In some embodiments, the non-mammalian source is jellyfish, marine invertebrate, fish, or amphibians.

[0244] In preferred embodiments of the fifth aspect, the peptide chain represented by X5is from a non-mammalian source. In some embodiments, non-mammalian source is jellyfish. In some embodiments, the jellyfish is from the sub-phylum Scyphozoa.

[0245] In certain embodiments of the fifth aspect, the peptide chain represented by X5is from the jellyfish sub-phylum Scyphozoa, wherein the Scyphozoa is selected from the group consisting of: the order Rhizostomeae, including, but not limited to, Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Cassiopea sp. (upside-down jellyfish), including but not limited to Cassiopea andromeda, the order Semaeostomease, including Aurelia sp., and other species such as Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, or any combination thereof. Preferably the source of the jellyfish collagen is Rhizostomas pulmo.

[0246] In some embodiments of the fifth aspect, the peptide chain represented by X5is selected from the group of proteins consisting of collagen, fibronectin, keratin, silk, resilin, laminin, and elastin.

[0247] In preferred embodiments of the fifth aspect, the peptide chain represented by X5is collagen. In some embodiments, the collagen is any one of the following: collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI, or XVII.

[0248] It will be understood that in embodiments of the fifth aspect wherein the peptide chain represented by X5is collagen, the modified protein according to the fifth aspect is a modified collagen, wherein the modified collagen comprises one or more groups of Formula V.

[0249] In some embodiments of the fifth aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-6 alkylene group, which Ci-6 alkylene group is optionally substituted with one or more X5agroups.

[0250] In preferred embodiments of the fifth aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, which Ci-4 alkylene group is optionally substituted with one or more X5agroups.

[0251] In some embodiments of the fifth aspect, each X5agroup is independently selected from the group consisting of -NH(R5b), and -OH, wherein each R5bis independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f.

[0252] In preferred embodiments of the fifth aspect, each R5bis independently a Ci-6 alkyl group, for example a Ci-4 alkyl group, such as a methyl group or an ethyl group, each optionally substituted with one or more groups selected from the group consisting of -N(R5d)(R5e), and =NR5f.

[0253] In more preferred embodiments of the fifth aspect, each R5bis a methyl group optionally substituted with one or more groups selected from the group consisting of -NH2, and =NH.

[0254] In certain embodiments of the fifth aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, which Ci-4 alkylene group is optionally substituted with one or more X5agroups, wherein each X5agroup is independently selected from the group consisting of -NH(R5b), and -OH, wherein each R5bis a methylene group optionally substituted with one or more groups selected from the group consisting of -NH2, and =NH.

[0255] In preferred embodiments of the second aspect, each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-4 alkylene group, which Ci-4 alkylene group is optionally substituted with one or more -OH groups.

[0256] In some embodiments of the fifth aspect, the modified protein comprising one or more groups of Formula V is a modified protein comprising one or more groups selected from the group of Formulae consisting of Formulae V-A, Formula V-B, Formula V-C, and Formula V-D: wherein:

[0257] X5is as defined above.

[0258] In certain embodiments of the fifth aspect, the modified protein comprising one or more groups of Formula V is a modified protein comprising one or more groups selected from the group of Formulae consisting of Formulae V-A, Formula V-B, and Formula V-C.

[0259] In some embodiments of the third to fifth aspects, the modified protein according to any one of the third to fifth aspects is incorporated into a polymer matrix.

[0260] In a sixth aspect of the invention, there is provided a composition comprising the modified protein according to any one of the third to fifth aspects.

[0261] In some embodiments of the sixth aspect, the composition further comprises a chelating agent. Preferably the chelating agent is ethylene diamine tetra acetic acid (EDTA).

[0262] In some embodiments of the sixth aspect, the composition further comprises EDTA which is present in the composition at a concentration of about 0.5 mM or more, at about 1.0 mM or more, at about 1.5 mM or more, at about 2.0 mM or more, at about 2.5 mM or more, at about 3.0 mM or more, at about 3.5 mM or more, at about 4.0 mM or more, at about 4.5 mM or more, at about 5.0 mM or more, at about 6.0 mM or more, at about 7.0 mM or more, at about 7.5 mM or more, at about 8.0 mM or more, at about 9.0 mM or more, at about 10 mM or more, at about 12 mM or more, at about 14 mM or more, at about 15 mM or more, at about 20 mM or more. In preferred embodiments of the sixth aspect, the composition further comprises EDTA at a concentration at about 2.0 mM or more, more preferably at about 3.0 mM or more, yet more preferably at about 5.0 mM or more, most preferably at about 10 mM or more.

[0263] In some embodiments of the sixth aspect, the composition further comprises EDTA at a concentration from about 0.1 to about 10 mM, from about 0.5 to about 9.5 mM, from about 1.0 to about 9.0 mM, from about 1.5 to about 8.5 mM, from about 2.0 to about 8.0 mM, from about 2.5 to about 7.5 mM, from about 3 to about 7.0 mM, from about 3.5 to about 6.5 mM, from about 4.0 to about 6.0 mM, from about 4.5 to about 5.5 mM. Preferably the composition comprises EDTA at a concentration from about 0.1 to about 10 mM.

[0264] In some embodiments of the sixth aspect, the composition is formulated as a film, a membrane, or hydrogel composite.

[0265] In preferred embodiments of the sixth aspect, the composition is formulated as a film, a membrane, or hydrogel composite, with a thickness in a range from about 5 to about 1000 pm.

[0266] In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising the modified protein or the composition according to any one of the third to sixth aspects, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0267] Depending on how the modified proteins of the invention are formulated and administered they can exert a wide variety of effects suitable for a variety of therapeutic and non- therapeutic applications. For instance, a wound dressing comprising a modified protein of the invention can provide an enhanced vasodilation effect with respect to blood flow control.

[0268] Endogenously produced nitric oxide is an important component of the body's natural defence mechanism. Depending on its concentration, NO exerts antimicrobial effects in two ways. At low concentrations, NO acts as a signalling molecule that promotes the growth and activity of immune cells. At high concentrations, such as during the respiratory burst of a neutrophil, NO covalently binds DNA, proteins and lipids, thereby inhibiting or killing target pathogens.

[0269] In an eight aspect of the invention, there is provided a modified protein, composition, pharmaceutical composition according to any one of the third to seventh aspects, which releases nitric oxide at physiological temperature.

[0270] When used herein, "physiological temperature" is 37°C (± 1°C), such as from 36.5 to 37.5°C.

[0271] In some embodiments of the eighth aspect, the concentration of nitric oxide released by the modified protein at physiological temperature is greater than the concentration of nitric oxide released by the same modified protein at a temperature below physiological temperature, such as about 20°C, about 15°C, about 10°C, about 5°C, about 0°C or about -5°C.

[0272] Such an effect advantageously allows for the modified protein, composition, or pharmaceutical composition of the invention to be stored for a period of days, weeks or months at temperatures below physiological temperature (for example, at about 5°C), before being activated for use by increasing the temperature to a physiological temperature. In some embodiments of the eight aspect, the modified protein, composition, or pharmaceutical composition releases nitric oxide for at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 24, at least about 48 or at least about 72 hours at physiological temperature. Preferably the modified protein, composition, or pharmaceutical composition releases nitric oxide for at least about 12 hours at physiological temperature, more preferably for at least about 18 hours at physiological temperature, most preferably for at least about 24 hours at physiological temperature.

[0273] In a ninth aspect of the invention, there is provided the modified protein, composition, or pharmaceutical composition according to any one of the third to eighth aspects, for use as a medicament.

[0274] In some embodiments of the ninth aspect, there is provided a wound dressing comprising the modified protein, the composition, or the pharmaceutical composition of the invention formulated such that it acts as an effective disinfection agent.

[0275] In some embodiments of the ninth aspect, there is provided a wound dressing comprising the modified protein, the composition, or the pharmaceutical composition of the invention formulated such that an antimicrobial wound dressing is provided. In preferred embodiments, the wound dressing exhibits antimicrobial properties against bacteria, yeasts, fungi and / or viruses, in a more preferred embodiment the wound dressing exhibits antimicrobial properties against bacteria, yeasts, and / or fungi, in yet a more preferred embodiment the wound dressing exhibits antimicrobial properties against bacteria.

[0276] In a tenth aspect of the invention, there is provided a modified protein, composition, or pharmaceutical composition according to any one of the third to eighth aspects, for use in wound healing, for use in tissue healing, for use in immune modulation, for use in orthopaedics, for use in vascular surgery, for use as a antimicrobial, for use in inhibiting biofilms at the wound surface, for use in regulating immune responses, for use in promoting vasodilation, for use as a vasodilation agent, for use as a disinfection agent, for use in new blood capillary growth, for use in tissue remodelling, for use in stimulating angiogenesis, or for use in promoting epidermal stem cell migration.

[0277] In preferred embodiments of the tenth aspect, there is provided the modified protein, composition, or pharmaceutical composition according to the invention, for use in wound healing, for use in tissue healing, or for use in immune modulation.

[0278] In an eleventh aspect of the invention, there is provided the non-therapeutic use of the modified protein, composition, or pharmaceutical composition according to the invention in a cosmetic product.

[0279] In some embodiments of the eleventh aspect, there is provided the non-therapeutic use of the modified protein, composition, or pharmaceutical composition according to the invention in a cosmetic product for application to the skin of a subject. In some embodiments, the subject is a human.

[0280] The term "about" as used herein when referring to a measurable value such as an amount of a compound, pH, time, temperature, and the like, refers to variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. It is contemplated that, at each instance, such terms may be replaced with the notation "± 10%", or the like (or by indicating a variance of a specific amount calculated based on the relevant value). It is also contemplated that, at each instance, such terms may be deleted.

[0281] The inventors of the present invention have surprisingly observed that the S-nitrosothiol groups in the one or more groups of Formula I, or the one or more groups of Formula V, in the modified proteins, the compositions and the pharmaceutical compositions of the invention are particularly stable. Specifically, S-nitrosothiol groups are retained for detection of NO after prolonged storage of the modified proteins of the invention.

[0282] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0283] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of".

[0284] The present invention will now be further described with reference to the below Examples and studies.

[0285] EXAMPLES

[0286] Example 1: Isolation of collagen from a jellyfish source

[0287] There are a number of methods of isolating or purifying proteins and such methods are well-known in the art. The exact method of isolation or purifying a protein for use in the method of the invention is not particularly limiting. Any method well-known and routinely used in the art could be adapted for the purpose of obtaining purified, or isolated, proteins.

[0288] Collagenous material can be purified from jellyfish by acid extraction, whereby different anatomical parts of the jellyfish are bathed in an acidic solution. "Bathing", or "bathe", refers to the process of incubating the jellyfish in the acid solution for a sufficient amount of time in order to liberate the collagen molecule. Collagen extraction from marine and bovine derived materials is known in the art. Such extraction is described in WO 2018 / 220396 and WO 2018 / 046920 both of which are incorporated herein by refrence.

[0289] In the Examples contained herein an acid soluble collagen source was obtained from a barrel jellyfish, Rhizostoma pulmo to yield a master liquid collagen solution of about 4 mg / ml.

[0290] Example 2: Synthesis of a modified collagen comprising one or more groups of Formula I

[0291] This process was carried out in two steps and can be readily scaled. The first step involved the formation of cystaminated collagen from the reaction of collagen with cystamine in the presence of an activating reagent.

[0292] In the second step after purification through diafiltration, the cystaminated collagen was reacted with TCEP, a disulfide cleaving reagent, and the resulting collagen-thiol was then immediately used for nitrosation without purification. This two-step synthesis is summarised in Scheme 2 below. NH a) Cystamine &;

[0293] -s'

[0294] Activating agent a) TCEP b) NaNO; b) Dialyse c) Dialyse

[0295] S tep l Step 2

[0296] Reaction of collagen with cystamine

[0297] 25 mL of sodium chloride (50 mM) was added to 1 L of the master liquid collagen solution. The pH of the resulting solution was measured and adjusted to a pH of 5 with dilute sodium hydroxide solution (20 mM) as required.

[0298] A 1.55g sample of cystamine dihydrochloride (6.88 mmol, Sigma) was dissolved in 10 mL of deionised water and added to the collagen solution, followed by a 1.2g sample of 4- (4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methyl-morpholiniumchloride (DMTMM) (4.34 mmol, Acros) in 10 mL of deionised water. Whilst stirring, the pH of the resulting solution was adjusted to pH 5 as required.

[0299] The collagen-cystamine mixture was left stirring for 18 hours at 5°C.

[0300] After 18 hours the mixture was dialysed (50-100 kDa) using a solution of acetic acid (20mM) containing sodium chloride (50 mM) to ensure complete removal of cystamine from the protein solution yielding a cystaminated collagen solution of about 4 mg / mL.

[0301] Reduction and nitrosation of the cvstaminated collagen

[0302] The pH of 1 L of the cystaminated collagen solution was adjusted to pH 3 using dilute hydrochloric acid (20 mM) as required.

[0303] A 0.65g sample of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (2.27 mmol, Biosynth) was dissolved in 5 mL of deionised water and added to the cystaminated collagen solution. The pH of the resulting solution was measured and adjusted to pH 3 as required.

[0304] The cystaminated collagen and TCEP mixture was then stirred under vacuum for 18 hours at 5°C. After 18 hours the vacuum was released, and the solution allowed to reach room temperature.

[0305] A 195 mg of sodium nitrite (2.82 mmol, Sigma) was dissolved in deionised water (5mL) and added to the above mixture. The pH of the solution was measured and adjusted to pH 3 as required. The solution was then stirred under vacuum for 18 hours at 5°C.

[0306] After 18 hours the solution was dialysed (50-100 kDa) using a solution of acetic acid (20 mM) containing sodium chloride (50 mM) until the dialysate effluent showed an absence of any residual sodium nitrite as analysed with the Griess colour test (approximately five times the volume of the solution was removed) to yield the final modified collagen solution. The sample was either kept as a liquid solution (of about 4mg / mL) or freeze-dried as required.

[0307] Fast Protein Liquid Chromatography (FPLC) analysis was performed using a Pharmacia Biosystems FPLC system fitted with a UV-Vis Dual Channel detector monitoring at 220 and 280 nm. The column used was a Sepharose Q High performance column (10 x 25 cm). The flow rate was 0.8 mL / min and a buffer system of Phosphate buffer pH 7.5 (50 mM) and Phosphate buffer pH 7.5 (50 mM + NaCI IM) was utilised alongside a linear gradient.

[0308] The resulting chromatograms from FPLC analysis on the master liquid collagen solution and the final modified collagen solution is shown in Figures 1A and IB respectively.

[0309] Example 3: Synthesis of a modified collagen comprising one or more groups of Formula V

[0310] This process was carried out in two steps and can be readily scaled as required. The first step involved the formation of a thiolated collagen from the reaction of collagen with 2- iminothiolane, more commonly referred to as Traut's reagent.

[0311] In the second step after purification through diafiltration, the thiolated collagen was nitrosated to yield the modified collagen comprising one or more groups of Formula V. This two-step synthesis is summarised in Scheme 3 below.

[0312] Scheme 3: Synthesis of Modified Protein Comprising S-nitrosothiol Groups using Traut's Reagent.

[0313] Reaction of collagen with Traut's reagent

[0314] To 100 mL of master liquid collagen solution a phosphate buffer solution (7.9 g of K2HPO4, 0.434 g of KH2PO4 in water made to 1 L) was added together with 59 mg of EDTA and 1.16 g of NaCI. The resulting solution was stirred to ensure complete dissolution. The pH of the resulting solution was measured and adjusted to pH 8 using a sodium hydroxide solution (1 M) as required.

[0315] 2-Imonothiolane, Traut's reagent, (200 pL, 30.3 mM), was then added to the above solution and left stirring for 24 hours at room temperature. After 24 hours the reaction was quenched by adding glycine powder (10 mM) and the pH of the solution immediately adjusted to pH 3 using a solution of hydrochloric acid (1 M) as required.

[0316] The resulting mixture was dialysed (50-100 kDa) using a solution of acetic acid (20 mM) containing sodium chloride (50 mM) until five times the volume of the solution was removed to yield a thiolated collagen solution of about 4 mg / mL. Nitrosation of the thiolated collagen

[0317] A 195 mg sample of sodium nitrite (2.82 mmol, Sigma) was dissolved in deionised water (5mL) and added to 1 L of the thiolated collagen solution. The pH of the solution was measured and adjusted to pH 3 as required using dilute hydrochloric acid (20 mM). The solution was then stirred under vacuum for 18 hours at 5°C.

[0318] After 18 hours the solution was dialysed (50-100 kDa) using a solution of acetic acid (20 mM) containing sodium chloride (50 mM) until the dialysate effluent showed absence of any residual sodium nitrite as analysed with the Griess colour test (approximately five times the volume of the solution was removed) to yield the final modified collagen solution.

[0319] The sample was either kept as a liquid solution (of about 4 mg / mL) or freeze-dried as required.

[0320] Example 4: Chemical analysis of modified collagen samples

[0321] Four modified collagen samples, each synthesised according to the synthetic method of Example 2 were analysed using spectrophotometric and nitric oxide (NO) measurement techniques. Figure 2 illustrates the results from absorption analysis between 300-382 nm. An absorption peak at ~345 nm was observed in all samples analysed. The peak was stable and was still visible several weeks after storage at ~5°C. However, S-NO bonds typically display an absorption peak at 330-340 nm, so further analysis was carried out to confirm the presence of an S-NO moiety.

[0322] By way of confirmation of the presence of an S-NO moiety in the modified collagen samples described herein, NO concentration measurements were taken using a chemiluminescent NO analyser. Sample ID's "Jell 02", "Jell 03", and "Jell 04" correspond to modified collagen samples synthesised according to Example 2, and each sample was stored as a liquid solution prior to analysis. Sample ID "06501" also corresponds to a modified collagen sample synthesised according to Example 2, however this sample was immediately freeze dried after purification (dialysis). Finally, sample ID "Comparative collagen 1" corresponds to a non-nitrosated modified collagen sample. "Comparative collagen 1" was synthesised according to Example 2, however the final nitrosation step was omitted (i.e. the addition of sodium nitrite was omitted). After purification by dialysis, "Comparative collagen 1" was immediately freeze dried.

[0323] In keeping with the spectrophotometric measurements, it was established that all modified collagen samples analysed, except for "Comparative collagen 1", released NO under stimulation from CuCl2 (100 pL, 200 nM) in the reaction chamber. The sample volume injected was 100 mL of a 1 in 10 dilution with distilled water. Figure 3 shows the resulting chemiluminescence trace of the NO signals detected and the concentration of NO is indicated below the baseline. Copper ions are known to selectively release NO from S- nitrosothiol groups, therefore this data confirms the effect of NO release from the modified collagen samples analysed to be S-NO mediated.

[0324] To investigate the stability of the S-NO groups in these modified collagen samples, these experiments were repeated again after storage of each sample for ten days at room temperature conditions. Table 1 below shows the concentration of nitric oxide (nM) detected for each sample analysed at day one and at day ten (dilution factor accounted for). Table 1: Comparison of [NO] at day 1 and at day 10 for modified collagen samples left at room temperature conditions.

[0325] Example 5: Nitrite assay

[0326] The following assay utilises the Griess Reagent which is a well-known protocol used for the estimation of nitrite concentration in a variety of mediums. The Griess Reagent reacts with any nitrite ions in solution, producing a pink-red dye which can be used to provide an indication as to the concentration of the nitrite. By assaying samples of modified proteins which have been stored for a period of time, the stability of the S-nitrothiol groups of the modified proteins was further investigated.

[0327] General Method A

[0328] Griess reagent (100 pL) was added to each sample (200 pL). The solution was left standing for 5 mins before being diluted with hydrochloric acid (20 mM, 2.2 mL) and left at room temperature for 1 hr. The absorption at 546 nm (A546) of each sample was subsequently recorded.

[0329] Example 5A: Sodium nitrite standards

[0330] General Method A was performed on standard samples of NaNOz (0.5, 1, 1.5 and 2 pg / mL). The A540 values of each sample are shown in Table 2 below and were used to generate a calibration curve which is shown in Figure 4A. Table 2: Absorption at 540 nm of standard sample of sodium nitrite

[0331] Example 5B: Modified collagen samples General Method A was then performed on modified collagen samples manufactured according to the synthetic method of Example 2, immediately after synthesis (day 0) and 26 days later (day 26). Between day 0 and day 26, each sample of modified collagen was left standing at room temperature as a liquid sample of approximately 4 mg / mL.

[0332] Sample ID "NN 03801" was also synthesised according to the synthetic method of Example 2, however during the reaction of collagen with cystamine, the activating agent EDC / NHS was used (instead of DMTMM).

[0333] The results are shown in Table 3 below, and are depicted graphically in Figure 4B.

[0334] Table 3 - Absorption at 546nm of various samples of S-nitroso modified collagen recorded at day 0 and at day 26.

[0335] Example 6: Effect of EDTA and NaCI on stability

[0336] From a batch of modified collagen manufactured according to the synthetic method of Example 2, aliquots were taken and kept at either 5°C or -20°C.

[0337] NaCI (50 mM) was added to sample ID "NN 5603" which was stored at 5°C. EDTA (10 mM equivalent) was added to sample ID "NN 5601" which was stored at 5°C and to sample ID "NN 5602" which was stored at -20°C. Sample ID "NN 5604" was stored at 5°C with no additives added.

[0338] At regular intervals, samples from each aliquot were taken and their A546 values were recorded according to General Method A. The results are shown in Figure 5 and show that the samples containing the chelating agent EDTA showed improved stability, through a larger absorbance at 546 nm.

[0339] The presence of the chelating agent stabilises the S-nitrosothiol groups of the modified collagen. Specifically, the presence of EDTA can eliminate the presence of transition metal ions, or other impurities that catalyse the decomposition of S-nitrosothiol groups. In contrast, the sample containing sodium chloride showed reduced stability relative to the sample that did not contain any sodium chloride or EDTA. Therefore, the presence of sodium chloride appears to stimulate the release of NO from the modified collagen. Example 7 - Effect of temperature on stability

[0340] From a batch of freeze-dried modified collagen, manufactured according to the synthetic method of Example 2, three samples were taken and kept at either 5°C, 20°C or 37°C for 24 hours.

[0341] After 24 hours, the A546 values were recorded for each sample according to General Method A. The results are provided in Figure 6 and show that after 24 hours the freeze- dried samples of modified collagen are still capable of releasing NO. Furthermore, the results show that NO release is promoted at elevated temperatures. Specifically, the absorbance at 546 nm for the modified collagen sample stored at physiological temperature (37°C) is approximately double the absorbance at 546 nm recorded in the other samples stored at lower temperatures.

[0342] Example 8 - In vitro efficacy testing

[0343] A vascular bioassay can be used to detect the presence NO with limits of detection in the low nM or even pM range. That assay can also be used as a confirmatory tool for the action of NO in the context of a biological tissue. Specifically, application of NO leads to relaxation of contracted vascular tissue, thus providing a read-out of the biological effect of that molecule.

[0344] Preliminary experiments were performed using a sample of modified collagen, manufactured according to the synthetic method of Example 2, which had been stored for three months at 5°C. A substantial relaxation (~75%) was observed upon exposure of pre-contracted rings of pig radial arterial rings to the modified collagen sample (see Figure 7).

[0345] The effect was entirely reversed by the addition of the selective soluble guanylate cyclase inhibitor, ODQ, providing further evidence that the effect is S-NO mediated.

[0346] Furthermore, the relaxation effect was sustained throughout a 2-hour exposure period, confirming a consistent and sustained release of NO from the three-month aged sample of modified collagen.

[0347] Comparative Example 9

[0348] A comparative sample of modified collagen comprising S-nitroso groups was synthesised from bovine collagen in accordance with the synthetic procedure detailed in WO 2016 / 166524, which is incorporated herein by reference. The UV absorption of the comparative sample of modified bovine collagen was monitored over a period of 90 minutes.

[0349] Prior to nitrosation, the comparative sample of modified bovine collagen was divided into two batches; one batch was stored as a liquid solution at room temperature with no additional additives; the other was stored as a liquid solution over ice. Sodium nitrite was then added to each sample to initiate nitrosation as detailed in WO 2016 / 166524.

[0350] As mentioned previously, typically S-NO bonds lead to specific absorption peaks in the range of 330-340 nm in the UV-visible spectrum. The comparative modified collagen samples synthesised according to WO 2016 / 166524 display an absorption peak at 335 nm. To investigate the stability of the S-NO groups in the comparative modified bovine collagen the absorption peak at 335 nm (A335) was monitored over a period of 90 minutes post nitrosation.

[0351] The results are shown in Figure 8 which shows a plot of log(A33s) vs time. The dashed horizontal line at log(A33s) ~ 0.3 corresponds to the half-life of the S-NO groups of each sample which had an initial log(A33s) ~ 0.6.

[0352] As can be seen from Figure 8, the comparative sample of modified bovine collagen is relatively unstable, with a half-life of approximately 20 minutes, which can be prolonged somewhat by cooling. In contrast, the modified collagen samples of the invention have been shown to exhibit improved S-NO group stability and are stable over a period of days (see, for instance, Figure 5 as discussed in respect of Example 6 above).

Claims

CLAIMS1. A method of producing a modified protein, the modified protein comprising one or more groups of Formula Iwherein :X1represents the peptide chain of the modified protein; each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group; each Rlais independently a Ci-io alkylene group, optionally substituted with one or more Xlagroups; each Xlagroup is independently selected from the group consisting of a Ci- io alkyl group, -C(O)ORlb, and -N(Rlc)(Rld), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)ORleand -N(Rlf)(R19); andRlb, Rlc, Rld, Rle, Rlfand R19, are each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group, the method comprising the steps of:(a) providing a protein comprising one or more groups of Formula II,II wherein :X1and L1are as defined above, and reacting the protein comprising one or more groups of Formula II with a compound of Formula III,Ill wherein:Rlais as defined above;R3ais selected from the group consisting of hydrogen and -SR3b;R3bis selected from the group consisting of hydrogen and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more X3agroups; each X3agroup is independently selected from the group consisting of a Ci-io alkyl group, -C(O)OR3c, and -N(R3d)(R3e), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)OR3fand -N(R39)(R3b); andR3c, R3d, R3e, R3f, R39and R3bare each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group, to provide a protein comprising one or more groups of Formula IV-A,wherein:X1, L1and Rlaare as defined above, or a protein comprising one or more groups of Formula IV-B,OX1- L1- U - N - R1a— S - SR3bT1V / “BD wherein:X1, L1, Rlaand R3bare as defined above;(b) optionally, wherein when step (a) provides a protein comprising one or more groups of Formula IV-B, providing a protein comprising one or more groups of Formula IV-A by reducing the S-S bond in the one or more groups of Formula IV-B to provide a -SH group; and(c) providing the modified protein comprising one or more groups of Formula I by nitrosating the -SH group of the protein comprising one or more groups of Formula IV-A.

2. The method according to claim 1, wherein the protein comprising one or more groups of Formula II is a protein comprising one or more groups selected from the Formulae consisting of Formula II-A, Formula II-B, and Formula II-C :OX1- H - OH TT.AOX11- CH2 - C 11 - OHII-B,wherein :X1is as defined in claim 1.

3. The method according to claim 1 or claim 2, wherein R3ais -SR3b.

4. The method according to claim 1 or 2, wherein R3ais hydrogen.

5. The method according to any one of claims 1 to claim 3, wherein R3bis a Ci-io alkyl group, optionally substituted with one or more X3agroups.

6. The method according to any one of the preceding claims, wherein each Xlagroup is independently selected from the group consisting of -C(O)OH and -NH2.

7. The method according to any one of claims 1 to 3, 5 or 6, wherein each X3agroup is independently selected from the group consisting of -C(O)OH and -NH2.

8. The method according to any one of claims 1 to 3, or 5 to 7, wherein the compound of Formula III is a compound selected from the group consisting of:

9. The method according to any one of claims 1, 2, 4, or 6 wherein the compound ofFormula III is a compound selected from the group consisting of:and10. The method according to any one of the previous claims, wherein step (b) comprises reacting a protein comprising one or more groups of Formula IV-B with a reducing agent, optionally wherein the reducing agent is selected from the group consisting of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT), and (2S)-2-amino-l,4-dimercaptobutane (DTBA).

11. The method according to any one of the previous claims, wherein step (c) is carried out on the unpurified product of step (b).

12. The method according to any one of the previous claims, wherein step (a) is performed in the presence of an activating agent, optionally wherein the activating agent is selected from the group consisting of 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)- 4-methyl-morpholinium chloride (DMTMM), 4-dimethylaminopyridine (DMAP), and a combination of l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

13. A method of producing a modified protein, the modified protein comprising one or more groups of Formula Vwherein:X5represents the peptide chain of the modified protein;each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group, which Ci-io alkylene group is optionally substituted with one or more X5agroups; each X5agroup is independently selected from the group consisting of - N(R5a)(R5b), and -OR5c;R5a, R5b, and R5care each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and = NR5f; andR5d, R5e, and R5fare each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, the method comprising the steps of:(d) providing a protein comprising one or more groups of Formula VI, x5- L5- NH2 VIwherein:X5and L5are as defined above, and reacting the protein comprising one or more groups of Formula VI with 2-iminothiolane (Traut's reagent) to provide a protein comprising one or more groups of Formula VII,wherein:X5and L5are as defined above; and(e) providing the modified protein comprising one or more groups of Formula V by nitrosating the -SH group of the protein comprising one or more groups of Formula VII.

14. The method according to claim 13, wherein the protein comprising one or more groups of Formula VI is a protein comprising one or more groups selected from the Formulae consisting of Formula VI-A, Formula VI-B, Formula VI-C, and Formula VI- D:X—NH2 VI-A.- wherein:X5is as defined in claim 13.

15. The method according to any one of the preceding claims, wherein nitrosating the - SH group of the protein comprising one or more groups of Formula IV-A in step (c), or nitrosating the -SH group of the protein comprising one or more groups of Formula VII in step (e), comprises reacting the protein with one or more reagentsselected from the group of reagents consisting of a solution of acidified nitrite, gaseous NO, and a S-nitrosothiol, preferably a solution of acidified nitrite.

16. The method according to any one of the preceding claims, wherein the method further comprises the step of:(i) Incorporating the modified protein into one or more of the groups comprising a collagen and a gelatin matrix; or(ii) Incorporating the modified protein into a polymer.

17. The method according to any one of the preceding claims, wherein the peptide chain represented by X1or X5is from natural or recombinant sources.

18. The method according to any one of the preceding claims, wherein the peptide chain represented by X1or X5is from a mammalian source, optionally wherein the mammalian source is cow, horse, pig, human, rat, sheep, or goat.

19. The method according to any one of claims 1 to 17, wherein the peptide chain of represented by X1or X5is from a non-mammalian source, optionally wherein the non-mammalian source is jellyfish, marine invertebrate, fish, or amphibians.

20. The method according to any one of the preceding claims, wherein the peptide chain represented by X1or X5is selected from the group of proteins consisting of collagen, fibronectin, keratin, silk, resilin, laminin, and elastin, preferably collagen, optionally wherein the collagen is any one of the following: collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI, or XVII.

21. A modified protein obtainable by a method according to any one of the preceding claims.

22. A modified protein, wherein the modified protein comprises one or more groups of Formula I:OX1- L1- (] - N - R1a— S - NO j wherein:X1represents the peptide chain of the modified protein; each L1is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group; each Rlais independently a Ci-io alkylene group, optionally substituted with one or more Xlagroups; each Xlagroup is independently selected from the group consisting of a Ci- 10 alkyl group, -C(O)ORlb, and -N(Rlc)(Rld), which Ci-io alkyl group is optionally substituted by one or more groups selected from the group consisting of -C(O)ORleand -N(Rlf)(R19); andRlb, Rlc, Rld, Rle, Rlfand R19, are each independently selected from the group consisting of hydrogen, a Ci-io alkyl group, and a C3-6 cycloalkyl group.

23. The modified protein of claim 22, wherein the modified protein comprising one or more groups of Formula I is a modified protein comprising one or more groups selected from the group of Formulae consisting of Formulae I-A, Formula I-B, and Formula I-C:wherein:X1, and Rlaare as defined in claim 22.

24. The modified protein of claim 23, wherein each Rlais ethylene, optionally substituted with a -C(O)OH group.

25. A modified protein, wherein the modified protein comprises one or more groups of Formula V:wherein:X5represents the peptide chain of the modified protein; each L5is a linker independently selected from the group consisting of a chemical bond, and a Ci-io alkylene group, which Ci-io alkylene group is optionally substituted with one or more X5agroups; each X5agroup is independently selected from the group consisting of - N(R5a)(R5b), and -OR5c;R5a, R5b, and R5care each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group, which Ci-io alkyl group is optionally substituted with one or more groups selected from the group consisting of - N(R5d)(R5e), and =NR5f; andR5d, R5e, and R5fare each independently selected from the group consisting of hydrogen, and a Ci-io alkyl group.

26. The modified protein of claim 25, wherein the modified protein comprising one or more groups of Formula V is a modified protein comprising one or more groups selected from the group of Formulae consisting of Formulae V-A, Formula V-B, Formula V-C, and Formula V-D:wherein:X5is as defined in claim 25.

27. The modified protein according to any one of claims 21 to 26, wherein the peptide chain represented by X1or X5is selected from the group of proteins consisting of collagen, fibronectin, keratin, silk, resilin, laminin, and elastin, preferably collagen, optionally wherein the collagen is any one of the following: collagen type 0, I, II, III, IV, V, VI, VII, IX, X, XI, or XVII.

28. A modified protein according to any one of claims 21 to 27, wherein the modified protein is incorporated into a polymer matrix.

29. A composition comprising the modified protein according to any one of claims 21 to 28, optionally wherein the composition is formulated as a film, a membrane, or a hydrogel composite.

30. The composition according to claim 29, wherein the composition further comprises a chelating agent, preferably wherein the chelating agent is EDTA, optionally wherein the EDTA is present at a concentration of from about 0.1 to about 10 mM.

31. A pharmaceutical composition comprising the modified protein or the composition according to any one of claims 21 to 30, and a pharmaceutically acceptable carrier, excipient, or diluent.

32. A modified protein, composition, or pharmaceutical composition according to any one of claims 21 to 31, wherein the modified protein, composition, or pharmaceutical composition releases nitric oxide at physiological temperature, preferably wherein the modified protein, composition, or pharmaceutical composition releases nitric oxide at physiological temperature for at least about 24 hours.

33. A modified protein, composition, or pharmaceutical composition according to any one of claims 21 to 32, for use as a medicament.

34. A modified protein, composition, or pharmaceutical composition according to any one of claims 21 to 32, for use in wound healing, for use in tissue healing or for use in immune modulation.

35. The non-therapeutic use of a modified protein, composition, or pharmaceutical composition according to any one of claims 21 to 32 in a cosmetic product.

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