Therapeutic peptides and methods of treatment using thereof

Abstract

The present invention relates to a method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject, comprising administering to the subject peptides derived from bovine lactoferrin, which target the conformationally active region of TNFR1 and inhibit its signaling. In particular, the peptide is FKCRRWQWRMKK (SEQ ID NO: 7) and the disease or condition that is associated with abnormal TNF / TNFR1 signaling is inflammation. Also disclosed are new peptides having improved potency to inhibit ligand induced NF-KB activation downstream of TNFR1 signaling. In a preferred embodiment, the new peptide is GKCLRWNWRFKK (SEQ ID NO: 3).

Description

[0001] Therapeutic peptides and methods of treatment using thereof

[0002] Field of Invention

[0003] The invention relates generally to human biology. In particular, the specification teaches a method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject.

[0004] Background

[0005] Tumor necrosis factor (TNF) is a signaling protein that is produced by the body’s immune system and is a critical regulator of inflammatory responses against harmful stimuli such as infections. However, excessive amount of TNF can cause autoimmune diseases and conditions such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, ankylosing spondylitis. It can also lead to chronic inflammation and tissue damage. Anti-TNF drugs, such as etanercept (Enbrel), adalimumab (Humira), infliximab (Remicade), are currently used in the clinic to treat TNF-related diseases. These drugs act by neutralizing or inhibiting the activity of TNF, thereby reducing inflammation and alleviating the symptoms associated with these diseases and conditions. However, many of these drugs are expensive and can induce dangerous side effects due to global TNF blockade. Hence, the specific targeting of TNF receptor 1 (TNFR1), rather than the cytokine itself, presents an alternative approach to inhibit downstream activation and attenuate inflammation.

[0006] It would be desirable to overcome or alleviate at least one of the above-described problems, or at least to provide a useful alternative.

[0007] Summary

[0008] Disclose herein is a method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject, wherein the method comprises administering to the subject a polypeptide comprising or consisting of the amino acid sequence of: X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1), wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional valiant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xc, to Xs are each any amino acid or a functional variant thereof.

[0009] Disclosed herein is a method of inhibiting inflammation in a subject, wherein the method comprises administering to the subject a polypeptide comprising or consisting of an amino acid sequence of:

[0010] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein X(, to Xs are each any amino acid or a functional variant thereof.

[0011] Disclosed herein is a method of inhibiting Tumor necrosis factor receptor 1 (TNFR1) signaling in a mammalian cell, the method comprises contacting the cell with a polypeptide comprising or consisting of an amino acid sequence of:

[0012] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional valiant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional valiant thereof, and wherein Xr, to Xs arc each any amino acid or a functional variant thereof.

[0013] Disclosed herein is a polypeptide comprising or consisting of the amino acid sequence of: X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xe to Xg are each any amino acid or a functional variant thereof.

[0014] Disclosed herein is a pharmaceutical composition comprising the polypeptide as defined herein.

[0015] Disclosed herein is a polynucleotide encoding the polypeptide as defined herein.

[0016] Disclosed herein is a vector comprising the polynucleotide as defined herein.

[0017] Disclosed herein is aa polypeptide or pharmaceutical composition as defined herein for use as a medicament.

[0018] Brief Description of Drawings

[0019] Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

[0020] Figure 1: FKC interacts with TNFR1 and induces receptor conformational change. (A) Schematic representation of the TNFR1 / TNF functional network consisting of TNFR1 dimers held together by TNF trimers (orange) upon ligand binding to form active signaling complex. PLAD: pre-ligand assembly domain, ECD: extracellular domain, and CD: cytosolic domain. (B) Schematic representation of the TNFR1 FRET biosensor with TNFR1ACD-GFP and TNFR1ACD-RFP used as the FRET pair. (C) Overexpression of the TNFR1 FRET biosensor in HEK293 cells leads to an efficient FRET observed in the donor-acceptor (indicated by DA) FRET pair as compared to the donor-only (indicated by D) no FRET control. (D-E) FKC reduces FRET of the TNFR1 biosensor in (D) a dose-dependent manner and (E) with an EC50 of 25 pM. (F) FKC does not cause any FRET change in the TNFR2 biosensor, which is acting as a control for receptor- specific interaction. (G) FKC is a 12-mer peptide (FKCRRWQWRMKK (SEQ ID NO: 7) that can induce TNFR1 conformational change. Data are means ± SD of N=4 independent experiments. *P<0.05, ***P<0.001, ****P<0.0001 and ns indicates non- significance by unpaired Student’s t test for comparison between two samples and one-way ANOVA with post hoc Tukey’s test for multiple comparisons.

[0021] Figure. 2. FKC inhibits TNFR1 signaling in a receptor-specific manner. (A) Western blotting analysis to characterize the effect of FKC in TNF induced TNFR1 downstream signaling pathways including IicBa degradation and NF-KB activation. (B- F) FKC inhibits TNF induced (B) phosphorylation of hcBa (plKBa), (C) IicBa degradation, (D) phosphorylation NF-KB component p65 (pp65) in HEK293 cells in a dose-dependent manner without affecting the levels of (E) NF-KB component p65 and (F) TNFR1. 0-actin was used as a loading control. (G) TNF stimulates NF-KB activation in HEK293 cells with wild-type (WT) TNFR1 but not in TNFR1 knockout (KO) cells. (H) FKC inhibits TNF induced NF-KB activation in HEK293 cells in a dose-dependent manner with an IC50 of 27 pM. (I) FKC docs not inhibit basal NF-KB activation in TNFR1 KO HEK293 cells. Data are means ± SD of N=4 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 and ns indicates non-significance by unpaired Student’s t test for comparison between two samples and one-way ANOVA with post hoc Tukey’s test for multiple comparisons.

[0022] Figure. 3. FKC attenuates inflammation in mice with intraperitoneal TNF injection. (A) TNF induces an increase in the plasma cytokines levels of TNF, MCP-1, IFN-y, IL- la, IL-ip, and IL-6 in both male and female mice, and FKC reduces all cytokine levels in a dose-dependent manner. (B-C) Immuno staining of liver, kidney, and lung tissues and image quantification illustrating the effect of FKC (40 mg / kg) in reducing macrophage activation in both male and female mice as char acterized through CD68-positive signals. (D-F) Western blotting analysis and quantification of liver, kidney, and lung tissues showing the effect of FKC (40 mg / kg) in inhibiting TNFR1 signaling in both male and female mice as characterized by changes in the level of downstream signaling molecules including phosphorylation of IxBa (plKBa), hcBa degradation, phosphorylation of NF-KB component p65 (pp65), p65, and TNFR1, with P-actin as a loading control. Representative images and blots shown were obtained from male mice. Data are means ± SD of N=12 mice (N=6 male mice and N=6 female mice) per treatment group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns indicates non-significance by one-way ANOVA with post hoc Tukey’s test for multiple comparisons.

[0023] Figure 4. FKC targets the conformationally active region of TNFR1 and perturbs receptor conformational dynamics. (A) Unbiased molecular dynamics (MD) simulations of FKC performed starting from the selected docked pose (Pose 4) from the HPEPDOCK program. The time evolution of FKC along the 250 ns simulation is indicated using a color gradient (blue-white-red) as indicated in the bar at the top of the panel. Two residues of FKC, Fl and R5, are indicated using the sticks representation. (B) The MD simulation results suggest that the FKC peptide is bound at the dimer interface where it established interactions with both TNFR1 monomers at residues near' the conformationally active region which involves the ligand binding loop. Important interactions with the TNFR1B (brown) and TNFR1E (cyan) are indicated by a green and purple dash lines, respectively. (C) Time evolution plots of distances (in A) linked to interactions between FKC and residues in both monomers, particularly R5-N136B (FKC-chain B), R9-E109E (FKC-chain E), and W6-Y106E (FKC-chain E). (D) The root-mean-square-fluctuation (RMSF) was calculated for the Ca atoms of the segment covering the CRD2 / 3 (C70 to NT 16) for the last 50 ns of the trajectory (200 to 250 ns). In the FKC bound system (cyan line), a reduction in the fluctuations is observed in the segment Y103 to Fl 15, relative to the system where the peptide is not present (grey line). (E) Total binding free energy between FKC and TNFR1 is -30.2 kcal / mol. The binding free energy per residue is also illustrated. (F) Co-immunoprecipitation (co-IP) characterization probing the interaction of His-tagged FKC with full-length TNFR1, different TNFR1 segments (ECD: extracellular domain, PLAD: pre -ligand assembly domain, and CD: cytosolic domain), TRADD, and full-length TNFR2. The presence of His-tagged FKC is illustrated by the His-tag antibody as a control.

[0024] Figure. 5. FKC alters TNFR1 conformational states without blocking ligand binding or disrupting receptor-receptor interaction. (A) Co-immunoprecipitation (co-IP) characterization between TNFR1 and TNF to determine whether FKC (50 pM) affects receptor-ligand interaction. (B) Native gel characterization of purified recombinant TNFR1 ECD in the presence of FKC (50 pM), zafirlukast (ZAF, 100 pM), or co-treatment of ZAF+FKC to test their effects in disrupting ECD-ECD or receptorreceptor interaction. (C) Treatment of FKC (50 pM), ZAF (100 pM), or co-treatment of ZAF+FKC to the PLAD FRET biosensor to test their effects in disrupting PLAD-PLAD interaction. (D) Treatment of FKC (50 pM), ZAF (100 pM), or co-treatment (ZAF+FKC) to the TNFR1 FRET biosensor to test whether FKC affects the FRET reduction induced by ZAF. (E-F) Distance distribution between the (E) CRD2 / 3 domains (CRD2 / 3-CRD2 / 3 distance) and (F) CRD4 domain (CRD4-CRD4 distance) of both receptor monomers is plotted for the TNFRB-TNFR1E dimer with FKC bound (top panel) and TNFRA-TNFR1D dimer without FKC (bottom panel). The numbers at each plot indicate the percentage of time that each system explores either distances smaller or greater than 30 A in (E) or 15 A in (F). The CRD2 / 3-CRD2 / 3 and CRD4- CRD4 distances from the original X-ray structure of the TNFR1 dimer (PDB: 1NCF) is indicated in green. (G) Co-lP characterization and quantification between TRADD, TNFR1 and TNF for samples with and without treatment of FKC (50 pM) in the presence of TNF and a no ligand control. The equal amount of His-taggcd TNF on the coated beads and endogenous TNFR1 are shown as pull-down controls. (H) The allosteric inhibition mechanism of FKC operates by binding to TNFR1 and perturbing receptor conformational dynamics without affecting receptor-ligand and receptorreceptor interactions. FKC bound receptor dimer adopts an open and inactive conformation that is unable to recruit downstream signaling molecules, resulting in nonfunctional receptor-ligand complexes that are incapable of signaling. Data are means + SD of N=4 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 and ns indicates non- significance by unpaired Student’s t test for comparison between two samples and one-way ANOVA with post hoc Tukey’s test for multiple comparisons.

[0025] Figure 6 Optimization of the lead peptide FKC sequence using mPARCE computational approach to obtain new peptide sequences with enhanced binding energy, (A) The binding site of the lead peptide FKC on TNFR1 dimer. (B) The peptide optimization strategy that has been adopted in improving the binding affinity of the lead peptide FKC. (C) The binding energy of the lead peptide FKC. (D) The binding energy of GKC which is a new peptide sequence derived from FKC. (E-H) GKM, YKM, VKM, and KML which are new peptide sequences derived from GKC. Another peptide. IKCRRWQWRMK (IKC), was also derived from FKC and the inventors were unable to obtain a calculation of its binding energy using the current approach.

[0026] Figure 7. Total binding energies of FKC and its peptide derivatives and the individual contribution of each residue in binding with TNFR1 dimer.

[0027] Figure 8. New peptides derived from FKC inhibits ligand induced NF-KB activation downstream of TNFR1 signaling with improved potency in HEK293 cells.

[0028] Figure 9. GKC induces TNFR1 conformational change and inhibits ligand induced receptor downstream signaling pathways including IKBU degradation and NF-KB activation in HEK293 cells.

[0029] Figure 10. GKC attenuates inflammation in mice with intraperitoneal TNF injection.

[0030] Detailed Description

[0031] The present specification teaches a method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject.

[0032] Disclosed herein is a method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject, wherein the method comprises administering to the subject a polypeptide comprising or consisting of the amino acid sequence of:

[0033] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1), wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, w'herein X5 is N, Q, or a functional variant thereof, and w'herein Xe to Xg are each any amino acid or a functional variant thereof. Without being bound by theory, the inventors have demonstrated that FKCRRWQWRMKK (SEQ ID NO: 7) (12 amino acids peptide) possesses antiinflammatory properties. The effect of the FKCRRWQWRMKK (SEQ ID NO: 7) peptide comes from targeting the novel conformationally active region of the tumor necrosis factor (TNF) receptor 1 (TNFR1). FKCRRWQWRMKK (SEQ ID NO: 7) (12 amino acids peptide) is part of the Bovine Lactoferricin (25 amino acids peptide) and both peptides are derived from Bovine Lactoferrin (708 amino acids protein). Both Bovine Lactoferricin (25 amino acids peptide) and Bovine Lactoferrin (708 amino acids protein) have been known to have antimicrobial, antiviral, antifungal, and antiinflammatory properties. Hence, FKCRRWQWRMKK (SEQ ID NO: 7) (12 amino acids peptide) can be used to target inflammation by targeting TNFR1 signaling. The inventors have also made additional changes to the peptide to arrive at GKCLRWNWRFKK (SEQ ID NO: 3) (12 amino acids peptide) which has improved properties.

[0034] The terms "protein" and "polypeptide" are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds. Polypeptides of less than about 10-20 amino acid residues are commonly referred to as "peptides." The polypeptides of the invention may comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a polypeptide by the cell in which the polypeptide is produced and will vary with the type of cell. Polypeptides are defined herein, in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0035] The terms “non-polar amino acids”, “polar amino acids”, “hydrophobic amino acids”, “positively charged amino acids” “negatively charged amino acids” are all used consistently with the prior art terminology. Each of these terms is well-known in the art and has been extensively described in numerous publications, including standard biochemistry text books, describing properties of amino acids which lead to their definition as polar, non-polar or acidic. In general, the non-polar amino acids may refer to glycine, alanine, valine, isoleucine, leucine and proline. The non-polar amino acids may also include aromatic non-polar amino acids such as phenylalanine, tryptophan and tyrosine. The neutral polar amino acids may refer to serine, threonine, cysteine, glutamine, asparagine and methionine. The negatively charged amino acids may refer to aspartic acid and glutamic acid. The positively charged amino acids may refer to lysine, histidine or arginine.

[0036] The term “functional variant” may refer to natural or chemically synthesized derivatives or analogues of an amino acid that is known to a person skilled in the art. A “functional variant” of an amino acid may have one or more modification(s) or variation(s) to its side chain moieties. For example, a side chain moiety of a D- or L- amino acid may have been modified to include a straight chain or branched, cyclic or non-cyclic, substituted or non-substitutcd, saturated or unsaturated, alkyl, aryl or aralyl moiety. A side chain of a D- or L-amino acid may have been modified to include reactive functional groups such as an azido or alkyne group. The term “functional variant” may also include, but is not limited to, amino acids that have been modified by addition of one or more sugar / carbohydrate moiety, oligosaccharide, or lipid groups. A “functional variant” may be incorporated in vivo into a polypeptide via techniques that are known in the art such as via chemical synthetic methods. Other methods such as by incorporation of the functional variant into a polypeptide via selective pressure incorporation in bacteria can also be used.

[0037] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

[0038] TABLE 1

[0039] AMINO ACID SUB-CLASSIFICATION

[0040] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isolcucinc or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 2 under the heading of exemplary and preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. TABLE 2

[0041] EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS

[0042] The polypeptide may have a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids.

[0043] In one embodiment, Xi is absent. In one embodiment, Xi is F or a functional variant thereof. In one embodiment, Xi is G or a functional variant thereof. In one embodiment, Xi is Y of a functional variant thereof. In one embodiment, Xi is A or a functional variant thereof. In one embodiment, Xi is V or a functional variant thereof. In one embodiment, Xi is W or a functional variant thereof.

[0044] In one embodiment, X2 is K or a functional variant thereof. In one embodiment, X3 is C or a functional variant thereof. In one embodiment, X3 is M or a functional variant thereof.

[0045] In one embodiment, X4 is L, or a functional variant thereof. In one embodiment, X4 is R, or a functional variant thereof. In one embodiment, X4 is I, or a functional variant thereof.

[0046] In one embodiment, X5 is N, or a functional variant thereof. In one embodiment, X5 is Q, or a functional variant thereof.

[0047] In one embodiment, Xe is F or a functional variant thereof. In one embodiment, Xe is M or a functional variant thereof.

[0048] In one embodiment, X7 is K, or a functional variant thereof.

[0049] In one embodiment, Xs is K, or a functional variant thereof.

[0050] In one embodiment, X5 is N, Xi is any amino acid or a functional variant thereof, or is absent, X2 to X4 are each any amino acid or a functional variant thereof and Xe to Xs are each any amino acid or a functional variant thereof.

[0051] In one embodiment, X5 is Q, Xi is any amino acid or a functional variant thereof, or is absent, X2 to X4 are each any amino acid or a functional variant thereof and Xe to Xs are each any amino acid or a functional variant thereof.

[0052] In one embodiment, X5 is Q, Xi is F, G, Y, A, V or W, or a functional variant thereof, or is absent, X2 is K, or a functional variant thereof, X3 is C or M, or a functional variant thereof, X4is L, R or I, or a functional var iant thereof, Xe is F or M or a functional var iant thereof, X7 and Xs arc each K, or a functional variant thereof.

[0053] In one embodiment, the polypeptide comprises or consists of the amino acid sequence of: XI-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1 ), wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xe to Xs are each any amino acid or a functional variant thereof. The R in position 5 of SEQ ID NO: 1 may be replaced by a functional variant of R. The W in position 6 of SEQ ID NO: 1 may be replaced by a functional variant of W. The W in position 8 of SEQ ID NO: 1 may be replaced by a functional variant of W. The R of position 9 of SEQ ID NO: 1 may be replaced by a functional variant of R.

[0054] In one embodiment, the polypeptide comprises or consists of an amino acid sequence of:

[0055] XI-X2-X3-X4-R-W-N-W-R-X6-X7-X8(SEQ ID NO: 2), wherein Xi is F, G, Y, A, V or W, or a functional variant thereof, or is absent, wherein X2 is K, or a functional variant thereof, wherein X3is C or M, or a functional variant thereof, wherein X4 is L, R or I, or a functional variant thereof, wherein Xe is F or M or a functional variant thereof, wherein X7 and Xs are each K, or a functional variant thereof.

[0056] In one embodiment, Xi is F, G or Y, or a functional variant thereof, or is absent, X2 is K, or a functional variant thereof, X3 is C or M, or a functional variant thereof, X4 is L or R, or a functional variant thereof, Xe is F or M or a functional variant thereof, X7 and Xs are each K, or a functional variant thereof.

[0057] In one embodiment, Xi is F, G or Y, or a functional variant thereof, or is absent, X2 is K, or a functional variant thereof, X3 is C or M, or a functional variant thereof, wherein X4 is L, or a functional variant thereof, wherein Xe is F or M or a functional variant thereof, wherein X7 and X8are each K, or a functional variant thereof.

[0058] In one embodiment, Xi is F, G or Y, or a functional variant thereof, or is absent, X2 is K, or a functional variant thereof, X3 is C or M, or a functional variant thereof, wherein X4 is L or R, or a functional variant thereof, wherein Xe is F or a functional variant thereof, wherein X7 and X8are each K, or a functional variant thereof. In one embodiment, Xi is any amino acid or a functional variant thereof, or is absent, X2is any amino acid or a functional variant thereof, X3 is any amino acid or a functional variant thereof, X4 is L, or a functional variant thereof, wherein X6 is F or a functional variant thereof, wherein X? and Xs are each K, or a functional variant thereof.

[0059] In one embodiment, Xi is any amino acid or a functional variant thereof, or is absent, X2is K or a functional variant thereof, X3 is any amino acid or a functional variant thereof, X4 is L, or a functional variant thereof, wherein Xe is F or a functional variant thereof, wherein X? and Xs are each K, or a functional variant thereof.

[0060] In one embodiment, Xi is any amino acid, X2is K, X3 is any amino acid, X4is L, wherein Xe is F, wherein X7 and Xs are each K.

[0061] In one embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 70% (or at least 80% or 90%) identity to an amino acid sequence of:

[0062] GKCLRWNWRFKK (SEQ ID NO: 3),

[0063] GKMLRWNWRFKK (SEQ ID NO: 4),

[0064] YKMLRWNWRFKK (SEQ ID NO: 5),

[0065] KMLRWNWRFKK (SEQ ID NO: 6), or YKMLRWNWRFKK (SEQ ID NO: 9).

[0066] In one embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 70% (or at least 80% or 90%) identity to an amino acid sequence of:

[0067] GKCLRWNWRFKK (SEQ ID NO: 3),

[0068] GKMLRWNWRFKK (SEQ ID NO: 4),

[0069] YKMLRWNWRFKK (SEQ ID NO: 5), or KMLRWNWRFKK (SEQ ID NO: 6).

[0070] The term "sequence identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (z.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Methods of aligning amino acid sequences are well known in the art. For example, bioinformatics or computer programs and alignment algorithms such as ClusterW may be used to determine the “% identity” between two amino acid sequences.

[0071] In one embodiment, the polypeptide comprises or consists of an amino acid sequence of:

[0072] GKCLRWNWRFKK (SEQ ID NO: 3), GKMLRWNWRFKK (SEQ ID NO: 4), YKMLRWNWRFKK (SEQ ID NO: 5), or KMLRWNWRFKK (SEQ ID NO: 6).

[0073] In one embodiment, the polypeptide comprises or consists of an amino acid sequence of:

[0074] FKCRRWQWRMKK (SEQ ID NO: 7).

[0075] In one embodiment, the polypeptide is an inhibitor of Tumor necrosis factor receptor 1 (TNFR1) signaling.

[0076] The polypeptide may bind specifically to the TNFR1 polypeptide.

[0077] The polypeptide may target the conformationally active region of TNFR1 by binding to the cysteine rich domains (CRD2 / 3).

[0078] The polypeptide may comprise a targeting sequence. The targeting sequence may be a sequence that direct the peptide to a cell or organ (i.e. cell-type specific or organ specific targeting sequence). The targeting sequence may, for example, direct the polypeptide to the brain (e.g. a blood brain barrier penetrating peptide). Such peptides are well known in the art. Examples of blood brain barrier penetrating peptides include angiopep-2, penetratin, TAT peptide, RVG peptide, glutathione or fragments thereof.

[0079] Provided herein is a polypeptide comprising or consisting of the amino acid sequence of:

[0080] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1), wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein X(, to Xs are each any amino acid or a functional variant thereof, for use in treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject.

[0081] Provided herein is the use of a polypeptide comprising or consisting of the amino acid sequence of:

[0082] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1), wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xr, to Xs are each any amino acid or a functional variant thereof, in the manufacture of a medicament for treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject.

[0083] Disclosed herein is a method of inhibiting inflammation in a subject, wherein the method comprises administering to the subject a polypeptide comprising or consisting of an amino acid sequence of:

[0084] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein X(, to Xs are each any amino acid or a functional variant thereof.

[0085] In one embodiment, there is provided a method of inhibiting inflammation in a subject, wherein the method comprises administering a polypeptide comprising or consisting of an amino acid sequence of at least 70% (or at least 80% or 90%) sequence identity to FKCRRWQWRMKK (SEQ ID NO: 7) to the subject. The polypeptide may comprise or consist of an amino acid sequence having an amino acid sequence of any one of SEQ ID NOs: 1 -7.

[0086] In one embodiment, there is provided a method of treating a disease or condition that is associated with inflammation in a subject, wherein the method comprises administering a polypeptide comprising or consisting of an amino acid sequence of at least 70% (or at least 80% or 90%) sequence identity to FKCRRWQWRMKK (SEQ ID NO: 7) to the subject. The polypeptide may comprise or consist of an amino acid sequence having an amino acid sequence of any one of SEQ ID NOs: 1-7.

[0087] Provided herein is a polypeptide comprising or consisting of an amino acid sequence of:

[0088] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xe to Xs are each any amino acid or a functional variant thereof, for use in inhibiting inflammation in a subject

[0089] In one embodiment, there is provided an amino acid sequence of at least 70% (or at least 80% or 90%) sequence identity to FKCRRWQWRMKK (SEQ ID NO: 7) for use in inhibiting inflammation in a subject. In one embodiment, there is provided an amino acid sequence of at least 70% (or at least 80% or 90%) sequence identity to FKCRRWQWRMKK (SEQ ID NO: 7) for use in treating a disease or condition that is associated with inflammation in a subject

[0090] Provided herein is the use of a polypeptide comprising or consisting of an amino acid sequence of:

[0091] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xe to arc each any amino acid or a functional variant thereof, in the manufacture of a medicament for inhibiting inflammation in a subject.

[0092] In one embodiment, there is provided the use of an amino acid sequence of at least 70% (or at least 80% or 90%) sequence identity to FKCRRWQWRMKK (SEQ ID NO: 7) in the manufacture of a medicament for inhibiting inflammation in a subject.

[0093] In one embodiment, there is provided an amino acid sequence of at least 70% (or at least 80% or 90%) sequence identity to FKCRRWQWRMKK (SEQ ID NO: 7) in the manufacture of a medicament for treating a disease or condition that is associated with inflammation in a subject.

[0094] Disclosed herein is a method of inhibiting Tumor necrosis factor receptor 1 (TNFR1) signaling in a mammalian cell, the method comprises contacting the cell with a polypeptide comprising or consisting of an amino acid sequence of:

[0095] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xg to Xs are each any amino acid or a functional variant thereof.

[0096] Provided herein is a polypeptide comprising or consisting of an amino acid sequence of:

[0097] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xg to Xs are each any amino acid or a functional variant thereof, for use in inhibiting Tumor necrosis factor receptor 1 (TNFR1) signaling in a mammalian cell.

[0098] Provided herein is the use of a polypeptide comprising or consisting of an amino acid sequence of:

[0099] X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xg to Xs are each any amino acid or a functional variant thereof, in the manufacture of a medicament for inhibiting Tumor necrosis factor receptor 1 (TNFR1) signaling in a mammalian cell.

[0100] Disclosed herein is an isolated polypeptide comprising or consisting of the amino acid sequence of:

[0101] Xi-X2-X3-X4-R-W-X5-W-R-Xg-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xg to Xs are each any amino acid or a functional variant thereof. The polypeptide may comprise or consist of an amino acid sequence of:

[0102] X1-X2-X3-X4-R-W-N-W-R-X6-X7-X8 (SEQ ID NO: 2), wherein Xi is F, G, Y, A, V or W, or a functional variant thereof, or is absent, wherein X2 is K, or a functional variant thereof, wherein X3 is C or M, or a functional variant thereof, wherein X4 is L, R or T, or a functional variant thereof, wherein Xe is F or M or a functional variant thereof, wherein X? and Xs are each K, or a functional variant thereof.

[0103] The polypeptide may comprise or consist of an amino acid sequence of:

[0104] GKCLRWNWRFKK (SEQ ID NO: 3), GKMLRWNWRFKK (SEQ ID NO: 4), YKMLRWNWRFKK (SEQ ID NO: 5), or KMLRWNWRFKK (SEQ ID NO: 6).

[0105] In one embodiment, the polypeptide is not FKCRRWQWRMKK (SEQ ID NO: 7).

[0106] Provided herein is a polynucleotide encoding the polypeptide as defined herein.

[0107] The term "nucleic acid" includes a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. The terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence” and “polynucleotide” are used interchangeably herein unless the context indicates otherwise.

[0108] The term “construct” refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.

[0109] In one embodiment, there is provided a construct comprising the polynucleotide as defined herein.

[0110] Provided herein is a vector comprising the polynucleotide as defined herein.

[0111] The nucleic acid construct may be, or may be comprised in, a vector. A “vector” as used herein is a nucleic acid used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the target cell. Such vectors may include a promoter sequence operably linked to the nucleic acid sequence to be expressed. A vector may also include a termination codon and expression enhancers. As used herein, the term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g., promoter and / or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. Where appropriate, the resulting transcript may then be translated into a desired polypeptide.

[0112] Any suitable vectors, promoters, enhancers and termination codons known in the ail may be used. Suitable vectors include, for example, viral vectors and recombinant plasmids. Vector selection and engineering are within those of skill in the art.

[0113] Provided herein is a pharmaceutical composition comprising the polypeptide as defined herein.

[0114] The polypeptide as defined herein may be in the form of a solid or liquid pharmaceutical composition. Pharmaceutical compositions can be formulated with a pharmaceutically acceptable carrier for administration to a subject. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water, saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch. magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of polypeptide together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0115] The term “pharmaceutically-acceptable” as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent or flavouring agent of a composition according to the present disclosure must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents and flavouring agents can be found in standard pharmaceutical texts, for example, Remington’s The Science and Practice of Pharmacy (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0116] The pharmaceutical composition may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, gels, creams, powders and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions may be administered intravenously, subcutaneously or intramuscularly. In some embodiments, the compositions arc in the form of injectable or infusible solutions. In some embodiments, the administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intranasal, topical or transdermal). Provided herein is a polypeptide or pharmaceutical composition as defined herein for use as a medicament.

[0117] The term “treating” includes remedying a disease state or symptoms, preventing the establishment of disease, or otherwise preventing, hindering, retar ding, or reversing the progression of disease or other undesirable symptoms in any way whatsoever.

[0118] The term “subject” as used throughout the specification is to be understood to mean a human or may be a domestic or companion animal. While it is particularly contemplated that the methods of the invention are for treatment of humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as primates, fclids, canids, bovids, and ungulates. The “subject” may include a person, a patient or individual, and may be of any age or gender. In one embodiment, the subject is a human.

[0119] In one embodiment, the disease or condition is inflammation, or a disease or condition associated with inflammation.

[0120] In one embodiment, the disease or condition is a TNF-related disease.

[0121] The peptides as defined herein may be used to treat or prevent a TNF-related disease. In this regard, the TNF-related disease may be any one selected from the group consisting of autoimmune diseases, inflammatory diseases, cardiovascular diseases, metabolic diseases, immune disorders, neurological diseases, ophthalmic diseases, skin diseases, psychiatric diseases, infectious diseases, and cancers, but is not limited thereto.

[0122] Specifically, the TNF-related disease may be any one selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, plaque psoriasis, juvenile plaque psoriasis, psoriatic arthritis, polyarticular juvenile idiopathic arthritis, Behcet's enteritis, ankylosing spondylitis, axial spondyloarthritis, juvenile enthesitis-related arthritis, polymyalgia rheumatica, multiple sclerosis, thyroiditis, delayed hypersensitivity. allergy, contact dermatitis, atopic dermatitis, systemic lupus erythematosus, systemic sclerosis, adult-onset Still's disease, asthma, autoimmune thyroid disorder, Sjogren's syndrome, Kawasaki disease, pancreatitis, nephritis, hepatitis, pneumonia, chronic obstructive pulmonary disease, otitis media, angioplasia nephritis, myelodysplastic syndrome, osteoarthritis, sarcoidosis, granuloma annulare, Wegener's granulomatosis, lupus, hemolytic uremic syndrome, arteriosclerosis, vasculitis, heart failure, stroke, myocardial infarction, myocardial ischemia-reperfusion injury, sexual dysfunction, obesity, hypertension, diabetes mellitus and diabetic complication, hyperlipidemia, preeclampsia, kidney disease, liver disease, kidney injury, liver injury, snake bite, allograft rejection, organ transplantation, graft versus host disease, dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pain, central nervous system disease, uveitis, Behcet's disease, diabetic macular edema, macular degeneration, orbitopathy, glaucoma, hidradcnitis suppurativa, multiccntric reticulohistiocytosis, pityriasis rubra pilaris, eosinophilic fascilitis, panniculitis, necrobiosis lipoidica diabeticorum, cicatricial pemphigoid, pyoderma gangrenosum, Sweet's syndrome, subcorneal pustular dermatosis, scleroderma, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, dermatomyositis, polymyositis, bullous dermatosis, erythema nodosum, alopecia, depressive disorder, bipolar disorder, anxiety disorder, tuberculosis, viral infection, bacterial infection, fungal infection, protozoan infection, cerebral malaria, sepsis, septic shock, prostate cancer, skin cancer, colorectal cancer, kidney cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, lymphoma, glioma, osteosarcoma, leukemia, multiple myeloma, and cachexia, but is not limited thereto.

[0123] The disease or condition may, for example, be rheumatoid arthritis, metabolic dysfunction associated steatotic liver disease (MASLD), diabetes, Alzheimer’s disease, Parkinson’s disease, or multiple sclerosis.

[0124] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. By “non-metastatic” is meant a cancer that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. The term "metastatic cancer" refers to cancer that has spread or is capable of spreading from one part of the body to another. Generally, a nonmetastaticcancer is any cancer that is a Stage 0, I, or II cancer, and occasionally a Stage III cancer. A metastatic cancer, on the other hand, is usually a stage IV cancer.

[0125] The term "cancer" includes but is not limited to, breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and / or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, Hodgkin's disease, nonHodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer or a combination thereof.

[0126] The term “administering” and variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing a pharmaceutically effective amount of polypeptide to an organism, or a surface by any appropriate means. The polypeptide may be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration. The route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, anal, vaginal) or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). The polypeptide can be administered alone or can be co-administered or sequentially administered with other treatments or therapies. Forms of administration may include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.

[0127] For oral administration, the formulation of the polypeptide may be presented as capsules, tablets, powders, granules, or as a suspension. The preparation may have conventional additives, such as lactose, mannitol, corn starch, or potato starch. The preparation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, com starch, or gelatins. Additionally, the preparation may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose. The preparation may be further presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. The preparation may be presented with lubricants, such as talc or magnesium stearate.

[0128] For intravenous, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included, as required.

[0129] For intranasal administration (e.g., nasal sprays) and / or pulmonary administration (administration by inhalation), formulations of the polypeptide, including aerosol formulations, may be prepared in accordance with procedures well known to persons of skill in the art. Aerosol formulations may comprise either solid particles or solutions (aqueous or non-aqueous). Nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.) and atomizers may be used to produce aerosols from solutions (e.g., using a solvent such as ethanol); metered-dose inhalers and dry-pow'der inhalers may be used to generate small-particle aerosols. The desired aerosol particle size can be obtained by employing any one of a number of methods known in the art, including, w'ithout limitation, jetmilling, spray drying, and critical -point condensation. The polypeptide as defined herein may be administered at a “pharmaceutically effective amount” to the patient in need thereof. The term "pharmaceutically effective amount” includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “pharmaceutically effective amount”. However, for any given case, an appropriate “pharmaceutically effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0130] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0131] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0132] Throughout this specification and the statements which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0133] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0134] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary' skill in the art to which this invention belongs.

[0136] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0137] EXAMPLES

[0138] Materials and Methods

[0139] Peptides synthesis

[0140] FKC (FKCRRWQWRMKK-NH2, SEQ ID NO: 7)), rhodamine conjugated FKC (Rho- FKCRRWQWRMKK-NH2, SEQ ID NO: 7), and IDR (VQRWLIVWRIRK-NH2, SEQ ID NO: 8) peptides were synthesized using solid phase F-moc chemistry by CPC Scientific, Inc. with a purity greater than 95%. The FKC structure was modified with the addition of an amine group to convert the COOH terminal to an amide. This is to protect FKC from proteolysis carried out by carboxypeptidases which confers improved stability and increases the half-time of the peptide inside cells and animals. Dynamic light scattering was conducted to measure whether the peptide forms aggregation at high concentrations using Brookhaven 90Plus Particle Size Analyzer.

[0141] Cell culture

[0142] The human embryonic kidney 293 (HEK293) cells (ATCC, Cat# CRL-1573) were cultured in phenol red-free Dulbecco’s modified Eagle medium with glutamine and sodium pyruvate (DMEM, Gibco Cat# 1 1965-092) supplemented with heat-inactivated 10% fetal bovine serum (FBS HI, Gibco Cat# A5256701), as well as penicillin- streptomycin (10,000 U / mL) (Thermofisher Scientific, Cat#15140122). Cell cultures were maintained in an incubator with 5% CO2 at 37°C (Nuaire NU-5831E CO2 Incubator). The TNFR1 KO HEK293 cell line (Ubigene, Cat# YKO-HS1133) was generated by CR1SPR-U technology (CR1SPR based) using small fragment knockout strategy removing exon 2, 3, 4 and 5 in the target coding region of TNFRSF1A and cultured as described above.

[0143] Animal studies

[0144] The inventors used both male and female C57BL / 6.I mice at 8-week-old for the treatment procedures. The mice were housed in a temperature-controlled room (25 °C) in virus-free facilities on a 12 h light / dark cycle (7:30 a.m. on / 7:30 p.m. off) and water ad libitum. All mice were maintained under specific pathogen-free conditions and treated with humane care. All procedures were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) animal use protocol A22058 from Nanyang Technological University, Singapore. In each animal experiment, mice were randomly allocated to receive intraperitoneal injections containing either saline, mouse TNF (MedChemExpress, Cat# HY-P7090), or FKC ith and without rhodamine conjugation at a total volume of 200 pL per injection. FKC was injected at 20 or 40 mg / kg in all mice and mouse TNF was used at 250 pg / kg following previously published protocol. For the treatment groups, mice (N=12 per group, 6 male and 6 female) were first intraperitoneally injected with first dose of either saline or FKC for 24 h, followed by subsequent intraperitoneal injection of a second dose of saline as negative control or mouse TNF for 4 h to induce peripheral inflammation. Mice were sacrificed by CO2 euthanasia and a blood sample from each mouse was collected via cardiac puncture into a BD Vacutainer Serum Tubes (Thermo Fisher Scientific, Cat# 12396929). Mouse tissues from the liver, kidneys and lungs were also collected for subsequent analysis.

[0145] Western blotting

[0146] HEK293 cells cultured in 6- well plates (Merck, Cat# CLS3506) at 0.5 million cells / well were treated with phosphate-buffered saline (PBS) (Sigma-Aldrich, Cat# D8537) as a negative control and respective doses of FKC (5-50 pM) for 2 h, followed by 30 min of human TNF (50 ng / mL) (MedChemExpress, Cat# HY-P7416) treatment at 37 °C. Both the cells and mouse tissues were lysed and homogenized respectively using radioimmunoprecipitation assay (RIP A) lysis buffer (Thermo Fisher Scientific, Cat# 89900) containing 1% Halt Protease and Phosphatase Inhibitor Cocktail (100X) (Thermofisher Scientific, Cat# 78440), followed by running in precast protein gels and conducting standard Western blotting procedures.

[0147] NF-KB activation assays

[0148] Both TNFR1 WT and TNFR1 KO HEK293 cells were transfected in 6-well plates with the dual luciferase reporters containing NF-KB response elements at 1 pg of Firefly luciferase reporter gene (Promega, Cat# E8491) and 0.1 pg of Renilla luciferase reporter gene (Promega, Cat# E6931 ) using Lipofectamine 3000 (Invitrogen, Cat# L3OOOO15). After 24 h of incubation, the transfected cells were dispensed into 96- well (15,000 cclls / wcll) white, solid-bottom plates (Greiner Bio-One North America, Cat# 655076) and incubated with PBS (negative control) or FKC (5-50 pM) in the presence (50 ng / mL) and absence of TNF and IL-10 (McdChcmExprcss, Cat# HY-P7028) for 18 h at 37 °C. Following incubation, a solution of 50 pL of Dual-Glo Luciferase Reagent (Promega, Cat# E2920) was added and kept at room temperature for 15 minutes. The Firefly luminescence was measured using a BioTek Synergy Hl Microplate Reader. Subsequently, 50 pL of Dual-Glo Stop & Gio Reagent (Promega, Cat# E2920) was added and kept at room temperature for 15 minutes, and Renilla luminescence was measured using a luminometer. For TRADD induced NF-KB activation in HEK293 cells, 1 pg of TRADD plasmid or control plasmid was co-transfected with the NF-KB dual luciferase reporter genes followed by same procedure as described above. The Firefly luciferase activities were normalized to the Renilla levels, and all data from NF-KB activation assays w'ere normalized to luciferase activity of cells with ligand treatment or TRADD overexpression.

[0149] Tissue immunostaining and image acquisition

[0150] Mouse livers, kidneys, and lungs were first fixed in 4% paraformaldehyde (PFA) in PBS solution (VWR, Cat# ALFAJ61899.AK) for 24 h, followed by overnight fixation in 30% sucrose solution (Sigma- Aldrich, Cat# SO389). The samples were then embedded in Tissue-Tek O.C.T compound (Sakura Finetek USA, Cat# 4583), using disposable base molds measuring 15x15 mm (Epredia, Cat# 58950) and cut into 20 pm thick frozen sections using a cryostat (Leica, Cat# CM 1950) and placed on Superfrost Plus Adhesion Microscopic glass slides (Epredia, Cat# J1800AMNZ). The frozen sections were blocked with 5% normal goat serum (NGS) (Thermo Fisher Scientific, Cat#31872) for 1 h at room temperature. Subsequently, the sections were incubated with primary and secondary antibodies before the fluorescent images were acquired using a slide scanner.

[0151] Construction of the TNFR1 / TNF complex system and peptide-protein blind docking

[0152] A detailed physiologically relevant model of a proposed functional form of the TNFR1 receptor bound to TNF was built as followed. The structure of the three TNFR 1 dimers (TNFR1A-TNFR1D, TNFR1B-TNFR1E and TNFR1C-TNFR1F) bound to a TNF trimcr (TNFX-TNFY-TNFZ) with a threefold symmetry was constructed using the available X-ray crystallography information (PDB:7KP7). The structure of the TNFR1 prc-ligand dimer was obtained from the available crystallographic information (PDB: 1NCF). To identify possible molecular poses of FKC in the TNFR1 / TNF system, the inventors utilized the HPEPDOCK 2.0 server which enables flexible peptide-protein docking by fast modeling of peptide conformations and global / local sampling of binding orientations. To reduce the processing time, only a dimeric structure (e.g., the TNFR1B- TNFR1E chains) of the TNFR1 system was utilized. Using the suggested default parameters, the program generated 10 different molecular poses for FKC. Docking of bovine lactoferricin (PDB : 1LFC), which is a segment of bovine lactoferrin (PDB : 1BLF) (71) on TNFR1 was performed using the same method. The amino acids sequences of FKC and bovine lactoferricin correspond to residues 36-47 and residues 36-60 of the bovine lactoferrin structure.

[0153] All-atom molecular dynamics (MD) simulations

[0154] Selected molecular poses obtained from the peptide-protein docking protocol were evaluated with all-atom MD simulations. The atomistic MD simulations were performed with the NAMD software using the CHARMM36 force field. The protein system was simulated at a temperature and pressure of 37 °C and 1 atm, respectively, and at a salt concentration of 0.15 M of NaCl. The entire systems are composed of approximately 215,000 atoms. The length of bond involving hydrogen atom were constraint to their equilibrium values using the SHAKE algorithm. The unbiased MD simulations were carried out using a 2.0 fs time step and the entire simulations consist of 250 ns.

[0155] FRET assays

[0156] The FRET biosensors were generated by transient transfection of HEK293 cells in 6- well plate with the respective donor-only and donor / acceptor FRET pair DNAs using Lipofectamine 3000. After the transfection, cells were transferred into 96-well plates (15,000 cells / well) followed by treatment with PBS (negative control), FKC (5-50 pM), or zafirlukast (ZAF, MedChemExpress, Cat#HY17491) for another 24 h followed by fluorescence intensity measurement.

[0157] Co-immunoprecipitation pull-down assays

[0158] To perform co-immunoprecipitation, Ni-NTA His-Tag Purification Agarose beads (MedChemExpress, Cat# HY-K0210) were first mixed with His-taggcd human TNF or His-tagged FKC and incubated for 4 h at 4 °C and the unbound His-tagged TNF or His- tagged FKC was then removed by washing the beads with PBS. His-tagged TNF was used to probe for receptor-ligand and TNFR1-TRADD interactions by pulling down the receptor. His-tagged FKC was used to probe for the interactions between FKC and full- length TNFR1, different TNFR1 domains, TRADD, or TNFR2. Immunoprecipitated samples were resolved using standard Western blotting procedures.

[0159] Statistical analysis

[0160] Statistical analyses were performed using the GraphPad Prism 9 software. Statistical analysis was conducted by using unpaired Student’s t test for difference between two conditions and one-way ANOVA with post hoc Tukey’s test for multiple comparisons. Statistical significance was determined by P<().()5 and indicated byi:7’<().()5. **P<0.01, ***P<0.001, ****P<0.0001 or ns for non- significance

[0161] Example 1

[0162] FKC interacts with TNFRl and induces receptor conformational change The inventors first examined whether the two peptides, FKC and IDR, interact with TNFR1 to impart their anti-inflammatory effects such as inhibition of NF-KB activation by testing their functions in a TNFR1 FRET biosensor formed by pre-ligand receptor dimers (Fig. IB). Overexpression of the TNFR1 donor-acceptor (DA) FRET pair in HEK293 cells illustrated efficient FRET compared to the donor-only (D) no FRET control (Fig. 1C). Importantly, FKC reduced FRET in the TNFR1 biosensor in a dosedependent manner (Fig. ID) with a half maximal effective concentration (EC50) of 25 pM (Fig. IE), indicating that FKC interacts with TNFR1 to induce conformational change. On the other hand, TDR did not cause any FRET change to the TNFR1 biosensor, suggesting that IDR does not induce TNFR1 conformational change and might be acting through alternative inhibitor}' mechanisms or signaling pathways to attenuate inflammation. As a critical control, the inventors also tested the effect of FKC in a TNFR2 FRET biosensor which showed efficient FRET in the pre-ligand dimeric form (result not shown). Treatment of FKC did not cause a FRET change in the TNFR2 biosensor (Fig. IF), further demonstrating the specific interaction of FKC with TNFR1. These data revealed that FKC, a 12-mer peptide (FKCRRWQWRMKK, SEQ ID NO: 7) derived from bovine lactoferricin, which is a segment of bovine lactoferrin, is able to induce TNFR1 conformational change (Fig. 1G). Moreover, molecular docking of bovine lactoferricin, a peptide with 25 amino acids (AA), on TNFR1 suggests that the first 12AA segment consisting entirely of FKC has a stronger binding free energy than the second 13AA segment (result not shown), further supporting the interaction of FKC with TNFRl.

[0163] FKC inhibits TNFR1 signaling in a receptor-specific manner

[0164] To investigate whether FKC can functionally inhibit TNFR1 signaling, the inventors examined its effect on receptor downstream pathways including IKBOI degradation and NF-KB activation (Fig. 2A). TNF not only induced phosphorylation of IKBCI (Fig. 2B) but also IKB a degradation (Fig. 2C) in HEK293 cells and FKC treatment inhibited these effects in a dose-dependent manner between 5-50 pM (Fig. 2B-C). Correspondingly, TNF increased the phosphorylation of p65 which were prevented by FKC (Fig. 2D), demonstrating the inhibitory effect of the peptide on TNFR1 downstream signaling. As important controls, the inventors showed that the expression levels of p65 (Fig. 2E) and TNFR1 (Fig. 2F) are not affected by FKC. A luciferase reporter assay was then used to measure NF-KB activity where cells were transfected with vectors containing the NF- KB response element and activation of NF-KB leads to increased luciferase signals. TNF stimulated NF-KB activation to 5-fold the basal level in HEK293 cells with endogenous wild type TNFR1 (TNFR1 WT) (Fig. 2G). FKC inhibited NF-KB activation in TNFR1 WT HEK293 cells in a dose-dependent manner with a half maximal inhibitory concentration (1C50) of 27 pM (Fig. 2H), which is consistent with the results observed in the TNFR1 FRET assay. To examine whether FKC requires TNFR1 for its inhibitory action, the effect of FKC in TNFR1 knockout (TNFR1 KO) HEK293 cells was tested. The TNFR1 KO cells have a basal NF-KB activity of -20% relative luciferase level, similar to that of the unstimulated TNFR1 WT cells. This has been suggested to be due to ligand-free constitutive signaling of other cytokine receptors (40), making it a good control to test if FKC affects other proteins that modulate NF-KB signaling. First, TNF did not stimulate NF-KB activation in TNFR1 KO cells (Fig. 2G), confirming the lack of TNFR1 signaling. Importantly, FKC did not attenuate the basal NF-KB activity in TNFR1 KO cells, indicating its specificity in inhibiting TNFR1 signaling (Fig. 21).

[0165] To further determine the specificity of FKC, its effect on TNFRl-associated death domain (TRADD) and interleukin-1 beta (IL- ip) induced NF-KB activation was tested. Being an essential adaptor protein recruited to TNFR1 upon ligand activation of the receptor, TRADD overexpression has been reported to induce NF-KB activation independent of receptor activation. On the other hand, IL- 10 is a cytokine that binds IL- 1 receptor to stimulate NF-KB activation . These assays examine whether FKC affects TNFR1 downstream signaling molecules or other receptors mediating alternative NF- KB signaling pathways, making them the most complete controls for specificity. Overexpression of TRADD in HEK293 cells led to a 6-fold increase in NF-KB activation compared to control without TRADD overexpression (results not shown) and FKC did not inhibit this activation. IL- 10 treatment stimulated NF-KB activation to 10-fold of the basal level and again FKC did not exhibit any inhibitory effect. Finally, it was confirmed that FKC does not have any toxic effect in HEK293 cells. These results illustrated that FKC requires TNFR1 for its activity and the inhibitory effects observed are not due to any indirect effect that FKC has on other downstream signaling molecules or cytokine receptors. Example 2

[0166] FKC attenuates inflammation in mice with intraperitoneal TNF injection

[0167] Having shown the specific interaction of FKC with TNFR1 and its receptor- specific inhibition of downstream signaling, the inventors investigated the effect of FKC in vivo by using a simple mouse model of inflammation with intraperitoneal TNF injection. First, TNF injection in both male and female mice induced a significant increase in TNF, MCP-1, IFN-y, IL- la, IL-ip, and IL-6 cytokine levels in mouse plasma compared to the saline control (Fig. 3A). FKC treatment (20 and 40 mg / kg) reduced all cytokine levels in a dose-dependent manner (Fig. 3A), indicating the effectiveness of FKC in reducing overall inflammation in mice. As a control, the inventors checked that there is no aggregation of the peptide at the highest dose as characterized by dynamic light scattering. The inventors then examined whether FKC affects macrophage activation in liver, kidney, and lung. The localization of FKC in all tissues was confirmed through the presence of rhodamine conjugated FKC (results not shown). In all organs, it was observed that TNF induced macrophage activation as shown by the increase in the CD68-positive cells and FKC (40 mg / kg) reduced macrophage activation (Fig. 3B-C), further demonstrating its effect in reducing inflammation. Finally, it was determined whether the effect of FKC arises from inhibiting TNFR1 signaling by probing the receptor downstream signaling molecules in different tissues. It was illustrated that TNF injection not only induced phosphorylation of IKBO. but also IKBO. degradation, together with phosphorylation of p65 in all tissues, and FKC treatment at 40 mg / kg inhibited these downstream signaling (Fig. 3D-F). These results confirmed that FKC is acting through inhibiting TNFR1 to attenuate inflammation in vivo and similar inhibitory effects were observed in both male and female mice.

[0168] Example 3

[0169] FKC targets the conformationally active region of TNFR1 and perturbs receptor conformational dynamics To examine the binding site of FKC, the inventors first utilized a computational based molecular docking approach to probe for the peptide interaction with TNFR1. First, a physiologically relevant model of the proposed functional complex of TNFR1 and TNF ligand was constructed, formed by three TNFR1 dimers bound to a TNF trimer with a threefold symmetry. The protein assembly is composed by the three dimers, namely TNFR1A-TNFR1D, TNFR1B-TNFR1E, and TNFR1C-TNFR1F, bound to a TNF trimer, namely TNFX-TNFY-TNFZ. By utilizing a single dimeric structure (TNFR1B- TNFR1E), 10 molecular poses of the FKC interacting with TNFR1 were obtained using the HPEPDOCK docking program. Based on higher-ranking docking scores, peptide interaction at the dimer interface, and binding locations at the druggable sites on the ECD of the TNFR1 dimer, including the PL AD, LBD, and the conformationally active region of the receptor involving cysteine rich domains (CRD) 2 / 3, three molecular poses of the docked FKC, Pose 1, Pose 4, and Pose 6 were selected for further evaluation using unbiased MD simulations to test their interacting strength and binding stability with TNFR1. Importantly, the only molecular pose that displayed stable interactions through the 250 ns long MD simulations was the Pose 4 with FKC bound in the vicinity of the CRD2 / 3, which is close to the conformationally active region of the receptor.

[0170] In the starting position of Pose 4 from the molecular docking, FKC established interactions mainly with residues in one of the receptor monomers (TNFR1E); however, as the MD simulation progresses, a repositioning of the peptide molecular pose induced the formation of interactions with residues in the other receptor monomer (TNFR1B) (Fig. 4A). From the time evolution of FKC along the 250 ns long MD simulation trajectory, it was observed that the N-terminal segment of FKC (l-FKCRRW-6) (SEQ ID NO: 10) explored conformations around a relatively confined space due to the formation of stable interaction with residues in both receptor monomers (Fig. 4A). While peptide residues Fl, K2 and W6 formed interactions with residues in TNFR1E (e.g., Fl-Fl 15E, K2-E147E, and W6-Y 106E), R5 strongly forms polar interactions with the carbonyl group from contiguous residues in TNFR1B (R5-N134B, R5-T135B, and R5-N136B) (Fig. 4B). This specific interaction appears to anchor the FKC conformations so that interaction involving residues at the C-terminal segment are subsequently stabilized, including Q7-Q130B, W8-K132B, R9-E109E, and KI 1 -D93B (Fig. 4B). Time evolution plots involving relevant interactions, R5-N136B, R9-E109E, and W6-Y106E are shown (Fig. 4C), where it is evident that even though these interactions were not present in the initial protein complex, they were formed and conserved along the duration of the unbiased MD simulation. As indicated, not only FKC interacts with residues from both receptor monomers, but the nature of the interactions is also very diverse, including charged-charged (R9-E109B), aromatic (W6- Y106E), cation-pi (W8-K132B), and polar (R5-N134B) interactions (Fig. 4B). Overall, the binding of FKC between the TNFR1B-TNFR1E dimer and its interaction with residues from both monomers appear to hold them together.

[0171] Furthermore, the molecular pose of FKC placed it in close proximity with the conformationally active region around TNFR1 residues 100-1 17 previously identified to play a pivotal role in the receptor signal transduction. Indeed, a segment of the FKC peptide interacted with residues in the conformationally active region (c.g., Y106E, E109E, and Fl 15E) that may disrupt the normal conformational changes of the domain and the concomitant receptor activation, explaining its antagonistic properties (Fig. 4B). To suggest the possible consequences of the FKC binding in this region, the inventors calculated the root-mean-square-fluctuation (RMSF) for the Ca atoms of the segment covering the CRD2 / 3 (segment C70 to N116) for the last 50 ns of the trajectory (200 to 250 ns). A reduction in the fluctuations in the FKC bound system was observed in segment Y 103 to Fl 15 (highlighted in the rectangular box), relative to the system where the peptide is not present (Fig. 4D). This region forms a P-hairpin and contains the cysteine residue, Cl 14, involved in a disulfide bridge (C98-C114). At the tip of the P- hairpin (residue E109), the most significant change in the fluctuation was observed. The results suggest that FKC binding perturbed the dynamics of a conformationally active region that is central to the ligand-induced signal transduction of the receptor by making it more rigid in space.

[0172] By taking the final conformation of the peptide-protein complex as a representative structure, it was further shown that the total binding free energy of FKC on TNFR1 ECD dimer is -30.2 kcal / mol (Fig. 4E), validating its strong interaction with the receptor. Using IDR as a control, it was shown that the total binding free energy of IDR on TNFR1 ECD dimer at a similar docked pose is -28.5 kcal / mol. Although both FKC and IDR showed similar binding energies against TNFR1 ECD dimer, it is likely that IDR adopts a position that is not affecting the conformationally active region of the receptor or that no longer strengthens the dimer formation at these loci, hence not inducing a FRET change in the TNFR1 FRET biosensor (results not shown). From the analysis of the molecular poses and the per residue contribution, it was proposed that residues R5, W6, and Q7 in FKC play an important role in the interaction with the TNFR1 dimer. The chemical nature of these residues is different in 1DR, suggesting that this peptide may interact well with TNFR1 but without stabilizing the dimeric system as it is proposed in the case of FKC.

[0173] Importantly, it was shown from docking of FKC on the TNFR2 ECD dimer with a similar pose that the total binding free energy is -6.5 kcal / mol, which is nearly 5-fold less than its interaction with the TNFR1 ECD dimer and consistent with the lack of FRET changes in the TNFR2 FRET biosensor induced by FKC (Fig. IF). Since HawkDock decomposes the residue contribution, it was observed that the main difference arises from various stabilizing interactions in the FKC / TNFR1 complex that are lost in the FKC / TNFR2 complex which could be attributed to the difference in the TNFR1 and TNFR2 sequence. For instance, W6 in FKC no longer establishes aromatic interactions in TNFR2 since in this position TNFR2 contains a Leu instead of Tyr. In case of position W8 in FKC, this residue forms a cation-pi interaction with a Lys residue in TNFR1, which is absent in the FKC / TNFR2 complex because TNFR2 contains a Thr residue. Position K2 in FKC forms a salt-bridge with a Gin residue in TNFR1; however, this interaction is lost since TNFR2 bears a Thr residue. Lastly, position R5 in FKC is flanked by a Leu residue of TNFR1 that establishes a non-polar interaction along their non-polar sidechain region. At this position, TNFR2 has an Arg that may create some charge-charge repulsion with R5 from FKC.

[0174] To confirm the binding and interaction of FKC with TNFR1, the inventors conducted co-immunoprecipitation (co-IP) of His-tagged FKC with different segments of TNFR1 that arc known to be stable, including full-length TNFR1, PLAD, ECD, and the cytosolic domain (CD), together with the downstream molecule TRADD and full-length TNFR2 as controls. It was shown that FKC binds to full-length TNFR1 and ECD, but not TNFR1 PLAD and CD nor TRADD and full-length TNFR2, confirming that FKC is binding to the region around TNFR1 CRD2-4 (Fig. 4F). Isolating the transmembrane domain and different CRDs other than the PLAD has not been shown and the proteins may not be stable. There is a large number of cysteine-cysteine interaction present in the CRDs which, if disrupted, may induce misfolding and aggregation of the receptor fragments which is not desirable. Hence, while the computational study illustrated the binding site of FKC to be at CRD2 / 3, there is a possibility that it might also be interacting with CRD4 or the transmembrane domain of TNFR1.

[0175] FKC acts on TNFR1 allosterically by altering receptor conformational states without blocking ligand binding or disrupting receptor-receptor interaction

[0176] To elucidate the inhibitory mechanism of FKC, it was determined whether the receptor conformational change induced by FKC as well as the associated TNFR1 FRET change observed affects receptor-ligand interaction or rcccptor-rcccptor interaction. First, coIP of TNFR1 and TNF showed the same amount of receptor pulled down in the absence and presence of FKC (50 pM), indicating that FKC did not block receptor-ligand interaction, despite binding around the LBD (Fig. 5A). An anti-TNF (etanercept) was used as a positive control in disrupting receptor-ligand interaction. It was also observed that FKC did not abolish ligand-ligand interaction through native gel characterization of purified TNF ligand treated with FKC (50 pM). The inventors then examined whether FKC disrupts receptor-receptor interaction in purified TNFR1 ECD proteins. It was found that the small molecule, zafirlukast (ZAF), but not FKC, disrupted TNFR1 ECD- ECD interaction or receptor dimerization (Fig. 5B). Interestingly, co-treatment of FKC (50 pM) prevented ZAF from disrupting TNFR1 ECD-ECD interaction (Fig. 5B).

[0177] FKC could be increasing the interaction of the two receptor monomers through either directly acting on the PLAD or stabilizing the receptor dimer to hold the individual monomers together. It is unlikely that they act on the PLAD as the co-IP results showed that FKC does not interact with TNFR1 PLAD (Fig. 4F). The inventors further tested the effect of FKC in a PLAD FRET biosensor which only contain the TNFR1 PLAD and is specifically designed to test the effect of modulators in modulating PLAD-PLAD interaction. Overexpression of PLAD FRET pair in HEK293 cells resulted in efficient FRET observed (Fig. 5C). FKC did not reduce FRET of the PLAD biosensor (Fig. 5C), confirming that it did not bind and interfere with PLAD-PLAD interaction. A positive control of ZAF reduced FRET in the PLAD biosensor and co-treatment with FKC (50 pM) did not affect the FRET reduction induced by ZAF (Fig. 5C). This indicates that FKC is not acting on the PLAD and hence unable to prevent ZAF from disrupting PLAD-PLAD interaction.

[0178] To test whether FKC is holding the individual receptor monomers together to prevent ZAF from disrupting ECD-ECD interaction or dissociating the dimers, the inventors tested the effect of ZAF and co-treatment of ZAF and FKC in the TNFR1 FRET biosensor. Treatment of ZAF, which is known to disrupt receptor-receptor interaction, reduced the TNFR1 FRET signal to a larger extent than FKC, probably due to the fact that it was dissociating the dimers (Fig. 5D). Importantly, co-treatment of ZAF and FKC (50 pM) decreased the extent of FRET reduction induced by ZAF to a level similar to the FRET alteration induced by FKC alone (Fig. 5D). This suggests that FKC, which binds at the interface of two TNFR1 monomers in a pre-ligand dimer pair, is able to hold the individual receptor monomers together and prevent ZAF from dissociating them. Hence, these data indicated that FKC is acting through perturbing receptor conformational dynamics to inhibit TNFR1 signaling, which is consistent with the MD simulation results.

[0179] To further confirm that FKC is indeed acting on TNFR1 through altering receptor conformational dynamics, the inventors measured the CRD2 / 3-CRD2 / 3 and CRD4- CRD4 distance distributions from the MD simulation data in the FKC bound TNFR1 dimer and compared it to the system in the absence of the FKC. To measure the CRD2 / 3- CRD2 / 3 and CRD4-CRD4 distances in the TNFR1 dimer, the inventors took the distance of the center of mass of the backbone atoms from the CRD2 / 3 (C70 to N116) and CDR4 (C 117 to S 159) domains between TNFR1B-TNFR1E dimer with FKC bound and the equivalent distance between TNFR1A-TNFR1D dimer without FKC binding. The distance distributions indicated that the TNFR1B-TNFR1E dimer with FKC bound explored CRD2 / 3-CRD2 / 3 distance with >30 A at 71.4% of the time (Fig. 5E, top) and CRD4-CRD4 distance with >15 A at 26.5% of the time (Fig. 5F, top). This was at a higher frequency relative to the TNFR1A-TNFR1D dimer without FKC binding where CRD2 / 3-CRD2 / 3 distance of >30 A occurred at 60.2% of the time (Fig. 5E, bottom) and CRD4-CRD4 distance of >15 A occurred only at 5.1% of the time (Fig. 5F, bottom). It was also observed that FKC disrupted the double glutamine interaction between the two receptor monomer chains, namely Q130B-Q133E and Q133B-Q130E, that were originally present in the pre-ligand dimer without FKC binding and this potentially leads to the opening of the receptor dimer. These data suggested that the conformational change induced by FKC led to an increased frequency in the opening of the CRD2 / 3 and CRD4 of the TNFR1 ECD dimer.

[0180] To explain how an alteration in the TNFR1 conformational states could dictate receptor function, it was hypothesized that receptor conformational states may determine its accessibility to the downstream signaling machinery, including the recruitment of the downstream signaling molecules such as TRADD. To test this, the inventors performed another co-IP experiment to determine the interactions between TNFR1 and TRADD under ligand stimulation in the absence and presence of FKC in HEK293 cells. Importantly, it was found that significantly more TRADD is pulled down with TNF stimulation and FKC (50 pM) significantly reduced the amount of TRADD pulled down under TNF stimulated condition, suggesting that FKC operates by inducing a conformational opening of TNFR 1 which impedes the recruitment of TRADD (Fig. 5G) .

[0181] Together, the results demonstrated that FKC inhibits TNFR1 signaling by allosterically altering receptor conformational states, including increasing the frequency in the opening of the CRD2 / 3 and CRD4, as well as inducing a conformational opening in the cytosolic regions of the receptor. Importantly, FKC exerts its inhibitory effect on TNFR1 without blocking ligand binding or disrupting receptor-receptor interaction, but through impeding the recruitment of receptor downstream signaling molecules (Fig. 5H). This makes FKC a truly allosteric or noncompetitive inhibitor of TNFR1.

Claims

CLAIMS1. A method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject, wherein the method comprises administering to the subject a polypeptide comprising or consisting of the amino acid sequence of:X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1), wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xe to Xs arc each any amino acid or a functional variant thereof.

2. The method of claim 1, wherein:Xi is any amino acid or a functional variant thereof, or is absent,X2 to X4 are each any amino acid or a functional variant thereof,X5 is N, andXe to Xg are each any amino acid or a functional variant thereof.

3. The method of claim 1, w'herein the polypeptide comprises or consists of an amino acid sequence of:X1-X2-X3-X4-R-W-N-W-R-X6-X7-X8 (SEQ ID NO: 2), wherein Xi is F, G, Y, A, V or W, or a functional variant thereof, or is absent, wherein X2 is K, or a functional variant thereof, wherein X3 is C or M, or a functional variant thereof, wherein X4 is L, R or I, or a functional variant thereof, wherein Xe is F or M or a functional variant thereof, wherein X7 and Xg are each K, or a functional variant thereof.

4. The method of any one of claims 1 to 3, wherein the polypeptide comprises or consists of an amino acid sequence of:GKCLRWNWRFKK (SEQ ID NO: 3), GKMLRWNWRFKK (SEQ ID NO: 4), YKMLRWNWRFKK (SEQ ID NO: 5), orKMLRWNWRFKK (SEQ ID NO: 6).

5. The method of claim 1, wherein the polypeptide comprises or consists of an amino acid sequence of:FKCRRWQWRMKK (SEQ ID NO: 7).

6. The method of any one of claims 1 to 5, wherein the subject is a human.

7. The method of any one of claims 1 to 6, w'herein the polypeptide is an inhibitor of Tumor necrosis factor receptor 1 (TNFR1) signaling.

8. The method of claim 7, wherein the polypeptide targets the conformationally active region of TNFR1 by binding to the cysteine rich domains (CRD2 / 3).

9. The method of any one of claims 1 to 8, wherein the disease or condition is inflammation or a disease or condition that is associated with inflammation.

10. The method of any one of claims 1 to 9, wherein the polypeptide comprises a targeting sequence.

11. A method of inhibiting inflammation in a subject, wherein the method comprises administering to the subject a polypeptide comprising or consisting of an amino acid sequence of:X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, andwherein X6 to Xs are each any amino acid or a functional variant thereof.

12. A method of inhibiting Tumor necrosis factor receptor 1 (TNFR1) signaling in a mammalian cell, the method comprises contacting the cell with a polypeptide comprising or consisting of an amino acid sequence of:X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein Xe to Xs are each any amino acid or a functional variant thereof.

13. A polypeptide comprising or consisting of the amino acid sequence of:X1-X2-X3-X4-R-W-X5-W-R-X6-X7-X8 (SEQ ID NO: 1) wherein Xi is any amino acid or a functional variant thereof, or is absent, wherein X2 to X4 are each any amino acid or a functional variant thereof, wherein X5 is N, Q, or a functional variant thereof, and wherein X<, to Xs are each any amino acid or a functional variant thereof.

14. The polypeptide of claim 13, whereinXj is any amino acid or a functional variant thereof, or is absent,X2 to X4 are each any amino acid or a functional variant thereof, X5 is N, andXe to Xs are each any amino acid or a functional variant thereof.

15. The polypeptide of claim 13 or 14, wherein the polypeptide comprises or consists of an amino acid sequence of:Xi-X2-X3-X4-R-W-N-W-R-X6-X7-X8(SEQ ID NO: 2),wherein Xi is F, G or Y, or a functional variant thereof, or is absent, wherein X2 is K, or a functional variant thereof, wherein X3 is C or M, or a functional variant thereof, wherein X4 is L or R, or a functional variant thereof, wherein Xe is F or M or a functional variant thereof, wherein X7 and Xs are each K, or a functional variant thereof.

16. The polypeptide of any one of claims 13 to 15, wherein the polypeptide comprises or consists of an amino acid sequence of: GKCLRWNWRFKK (SEQ ID NO: 3),GKMLRWNWRFKK (SEQ ID NO: 4), YKMLRWNWRFKK (SEQ ID NO: 5), or KMLRWNWRFKK (SEQ ID NO: 6).

17. The polypeptide of claim 13, wherein the polypeptide is not FKCRRWQWRMKK (SEQ ID NO: 7).

18. A pharmaceutical composition comprising the polypeptide of any one of claims 13 to 17.

19. A polynucleotide encoding the polypeptide of any one of claims 13 to 17.

20. A vector comprising the polynucleotide of claim 19.

21. A polypeptide of any one of clams 13 to 17 or pharmaceutical composition of claim 18 for use as a medicament.

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