WO2018044234A1 - Cysteine-rich polypeptides and conjugates and methods of using the same - Google Patents

Abstract

The present invention relates to the identification and utilization of a novel class of cystine-rich peptides, wherein the peptide comprises three disulfide bridges that form a cystine-knot structural motif. Provided are polypeptides and conjugates as defined herein as well as methods of inhibiting neutrophil elastase, methods of delivering an agent of interest to a mitochondrion of a target cell, and methods of treating or preventing a disease, disorder, or condition in a subject using said polypeptides or conjugates.

Description

CYSTEINE-RICH POLYPEPTIDES AND CONJUGATES AND METHODS OF USING THE

SAME

CROSS-REFERENCE TO RELATED APPLICATION

This application makes reference to and claims the benefit of priority of the Singapore Patent Application Nos. 1 0201607254W and 10201607237U, both filed on 31 August 2016, the content of which is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

The present invention relates generally to cysteine-rich polypeptides and conjugates and methods of using said polypeptides and conjugates.

BACKGROUND OF THE INVENTION

Plant-derived peptides represent a promising group of natural products in drug discovery, as they fulfill the neglected chemical space between small-molecule metabolites (<1 kDa) and proteins (>8 kDa). However, these peptides have not received much attention as putative active compounds in medicinal plants and in drug development. Within the chemical space of 2-8 kDa, cysteine-rich peptides, which possess multiple disulfide bridges to enhance both structural and physical stabilities, fulfill the criteria of putative bioactive peptides in medicinal plants.

Plant knottins are cysteine-rich peptides and are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Therefore, there is still need in the art for novel plant knottins that are biologically active.

SUMMARY OF THE INVENTION

The present invention satisfies the aforementioned need in the art by providing the cysteine-rich polypeptides as described below.

In a first aspect, the invention relates to a polypeptide comprising or consisting of a peptide having the amino acid sequence of formula: (Xaa)m-Cys-(Xaa)n-Cys-(Xaa)p-Cys-Cys-(Xaa)q-Cys-(Xaa)i-Cys-(Xaa)j, wherein Xaa is any amino acid, preferably any naturally occurring amino acid, wherein independently m is 0 or 1 , n is an integer from 6-9, p is an integer from 2-7, q is 3 or 4, i is an integer from 4-13, and j is an integer from 1 -6, and wherein the peptide comprises zero, one, or more intramolecular disulfide bridges.

In various embodiments, the polypeptide comprises or consists of

(i) a peptide having the amino acid sequence set forth in any one of SEQ ID NOs:1 -8; or (ii) a peptide having the amino acid sequence sharing at least 65%, preferably at least 75%, even more preferably at least 85%, most preferably at least 95% sequence identity with the peptide of (i) over its entire sequence, wherein the six cysteine residues in the formula of claim 1 are invariable. In preferred embodiments, the polypeptide has the amino acid sequence set forth in SEQ ID NO:1 .

In various embodiments, the polypeptide comprises three disulfide bridges that form a cystine- knot structural motif.

In various embodiments, the polypeptide has a length of at least 22 preferably up to 46, most preferably between 27-39 amino acids.

In various embodiments, the polypeptide has a molecular weight of at least 2, preferably up to 8, most preferably between 2-5 kDa.

In a second aspect, the invention relates to a conjugate comprising or consisting of the polypeptide described herein and at least one agent of interest. In various embodiments, the at least one agent of interest is a therapeutic agent, a diagnostic agent, or a targeting agent.

In preferred embodiments, the at least one agent of interest is conjugated to the N-terminus of the polypeptide.

In various embodiments, the conjugate further comprises a linker connecting the at least one agent of interest to the polypeptide. In a third aspect, the invention relates to a method of inhibiting neutrophil elastase, the method comprising contacting said elastase with the polypeptide or conjugate described herein.

In a fourth aspect, the invention relates to a method of delivering an agent of interest to a mitochondrion of a target cell, the method comprising the steps of:

(a) providing a conjugate described herein, wherein the agent of interest is conjugated to the polypeptide described herein; and

(b) contacting the target cell with the conjugate or a composition comprising said conjugate. In a fifth aspect, the invention relates to a method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to said subject a therapeutically effective amount of the polypeptide or conjugate described herein, or a composition comprising said peptide or conjugate. In certain preferred embodiments, the disease, disorder, or condition is associated with neutrophil elastase, preferably an airway inflammatory disease selected from the group consisting of cystic fibrosis, asthma, COPD, and pulmonary emphysema.

In certain preferred embodiments, the disease, disorder, or condition is associated with altered mitochondrial function, preferably bioenergetic aging.

In certain preferred embodiments, the disease, disorder, or condition is a cancer.

In various embodiments, the subject is a mammal, preferentially a human.

In a six aspect, the invention relates to the use of the polypeptide or conjugate described herein as an inhibitor of neutrophil elastase, preferably human neutrophil elastase (SEQ ID NO:9).

In a seventh aspect, the invention relates to the use of the polypeptide or conjugate described herein as a mitochondria-selective targeting agent.

In a final aspect, the invention relates to the use of the polypeptide or conjugate described herein as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

Figure 1 : The mass spectrometry profiling of the aqueous extracts of Hibiscus sabdariffa revealed the presence of strong signals in the mass range of 2-4 kDa in the calyces, capsules and flowers. The strong signal of 2620 Da was designated as roseltide rT1 . (A) calyces, (B) capsule, (C) seed, (D) leaves, and (E) flower of Hibiscus sabdariffa were collected and profiled using Maldi-TOF MS.

Figure 2: De novo sequencing of roseltide rT1 . (A) S-alkylated roseltide rT1 was digested with trypsin, resulting in two tryptic fragments with the m/z of 545 and 2442; (B) MS/MS spectra of 545 Da fragment; (C) MS/MS spectra of 2442 Da fragment; (D) S-alkylated roseltide rT1 was digested with chymotrypsin at two sites, resulting in fragments with the m/z of 1 045 and 1328. The third peptide fragment of m/z 1 640 could not be detected ; (E) MS/MS spectra of 1045 Da fragment; and (F) MS/MS spectra of 1328 Da fragment. Figure 3: Roseltide transcripts from Hibiscus sabdariffa. The histogram depicts conservation among the putative amino acid sequences of roseltides by amino acid property grouping as determined by Jalview software. AEP: asparagine endopeptidase; PDI : protein disulfide isomerase; SPase: signal peptidase. Figure 4: (A) Sequence alignment of CKAI with roseltide rT1 ; allotide Ac2 from Allamanda cathartica; wrightide Wr-AI1 from Wrightia religiosa; alstotides As1 , As3 and As4 from Alstonia scholaris (B) Solution structure of roseltide rT1 ; (C) Superimposition of roseltide rT1 (black) on wrightide Wr-AI1 (PDB entry 2MAU) (grey) and alstotides As1 (PDB entry 2MM6) (grey).

Figure 5: (A) Acid, (B) human serum, (C) trypsin, and (D) pepsin stability of roseltide rT1 . All results were expressed as mean ± S. E.M. (n = 3).

Figure 6: Effects of roseltide rT1 on (A) Huh7 and (B) A549 cells. All results were expressed as mean ± S.E.M. (n = 3). ^*P < 0.05 compared to control group.

Figure 7: The effects of roseltide rT1 on human neutrophil elastase (SEQ ID NO:9). (A) The effects of different concentrations of roseltide rT1 or synthetic elastase inhibitor, N- methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, on human neutrophil elastase (HNE) activity was measured at 405 nm at 37 °C using N-methoxysuccinyl-Ala-Ala-Pro-Val p- nitroanilide as a substrate. All results were expressed as mean ± S.E.M. (n = 3); (B) MS spectra showed biotinylation of roseltide rT1 ; (C) The effects of biotin-rT1 on HNE activity. All results were expressed as mean ± S.E.M. (n = 3). ^*P < 0.05 compared to control ; (D) Silver-stained SDS-PAGE of the pull-down complex between HNE and biotin-rT1 . The leftmost lane shows a protein marker (Bio-rad, US) ; HNE-only lane: purified HNE only; biotin lane: purified HNE incubated with biotin and NeutrAvidin resin (control), biotin-rT1 lane: purified HNE incubated with biotin-rT1 and NeutrAvidin resin. The full-length gel image is provided in Figure 38.

Figure 8: Roseltide rT1 ameliorated human neutrophil elastase (HNE)-stimulated cAMP accumulation in CHO-K1 cells co-expressed with PAR2 receptor and Glosensor cAMP biosensor constructs. Comparison of PAR2 receptor expressions in cAMP-CHO cells using (A) confocal microscopy and (B) flow cytometry; (C) Effects of cAMP activator Forskolin (10 μΜ) on cAMP accumulation in PAR2-cAMP-CHO cells; (D) Effects of HNE with or without roseltide rT1 on cAMP accumulation in PAR2-cAMP-CHO cells. All results were expressed as mean ± S.E.M. (n = 3). ^*P < 0.05 compared to HNE-treated group. Figure 9: (A) Modeling the interaction between roseltide rT1 and the human neutrophil elastase (PDB entry: 1 HNE) using the ClusPro Version 2.0 server. (B) Interactions between roseltide rT1 and peptide chloromethyl ketone inhibitor (AAPA-CMK) (PDB entry: 1 HNE) with the catalytic triad of human neutrophil elastase formed by His57, Asp1 02, and Ser1 95. Figure 1 0: Site-specific N-terminal fluorescence labeling of roseltide rT1 .

Figure 1 1 : Quantitation of cellular uptake of Cy3-rT1 using flow cytometry in WI-38 human diploid lung fibroblast cells. Figure 12: Quantitation of cellular uptake of Cy3-rT1 using flow cytometry in HUVEC-CS cells.

Figure 13: Cell-penetrating properties of Cy3-rT1 by confocal microscopy. (A) The orthogonal view and (B) 3D rendering of the Z-stack images. Figure 14: Cellular uptake of Cy3-rT1 is not significantly affected by serum in HUVEC-CS cells.

Figure 1 5: Effect of temperature on the cellular uptake of Cy3-rT1 in WI-38 cells.

Figure 16: Effect of temperature on the cellular uptake of Cy3-rT1 in HUVEC-CS cells.

Figure 1 7: Effect of endocytosis inhibitor on the cellular uptake of Cy3-rT1 in HUVEC-CS cells. Figure 1 8: Cellular uptake of Cy3-rT1 in CHO-K1 (wild-type) and PgSA-745 (glycosaminoglycan deficient) cells.

Figure 1 9: Co-localization of Cy3-rT1 with mitochondria by confocal microscopy. Figure 20: Subcellular fractionation of HUVEC-CS cells after incubation with Biotin-rT1 . Figure 21 : Biotin-rT1 interacts with TOM20 using pull-down assay. Figure 22: Co-localization of Cy3-rT1 with TOM20-GFP by confocal microscopy.

Figure 23: In silico modelling of roseltide rT1 with TOM20.

Figure 24: (A) Roseltide rT1 induced mitochondrial membrane hyperpolarization and (B) increased mitochondria ROS level in isolated intact mitochondria from HUVEC-CS cells.

Figure 25: Roseltide rT1 increased cellular ATP levels in C2C12 cells.

Figure 26: MS/MS spectra of ATP50 fragment pulled-down by biotin-rT1 . Figure 27: (A) Primary sequence of ATP50 fragment pulled-down by biotin-rT1 . (B) Schematic structure of F-type ATP synthase. (C) Western blot results showed that roseltide rT1 interacts with ATP50. (D) The predicted structure of ATP50 was adopted from MODBASE based on the crystal structure of bovine ATP synthase (PDB entry: 2wssS). (D) In silico modelling of roseltide rT1 with ATP50.

Figure 28: Reduction and alkylation of Roseltide rT1 .

Figure 29: (A) HPLC-DAD chromatogram of aqueous extracts of the calyces of Hibiscus sabdariffa. (B) HPLC-DAD chromatogram and (C) MS spectrum of purified roseltide rT1 .

Figure 30: Hibiscus sabdariffa transcript database search for roseltides using PEAKS software.

Figure 31 : Chemical shift assignment of ¹ H, ¹ H-NOESY spectrum of roseltide rT1 . (A) The assignments of the NOE cross peaks between side chain protons and amide protons are displayed. (B) The assignments of the NOE cross peaks between amide protons are displayed.

Figure 32: NOE cross peaks between HNi and Ha, Hai-i . Figure 33: Structure and disulfide connection of rT1 . (A) The 20 best structures generated by CNSsolve 1 .3 with the disulfide bonds imposed are displayed in ribbon representation. (B) The three disulfide bonds, Cys1 -Cys16, Cys8-Cys21 and Cys 15-Cys26, are highlighted in light grey. The proton Ηβΐ and Ηβ2 are displayed in grey. (C) The 20 best structures generated by CNSsolve 1 .3 assuming all the cysteines reduced are displayed in ribbon representation. (D) The structure generated by CNSsolve 1 .3 without disulfide bonds imposed is superimposed to the structure with disulfide bonds imposed Cys1 -Cys16, Cys8-Cys21 and Cys 15-Cys26, in which the six cysteines are reduced and oxidized respectively. Figure 34: Chemical shifts of the protons of roseltide rT1 determined using ¹ H, ¹ H- TOCSY and NOESY.

Figure 35: NOE cross peak between the Hps of the two cysteines in each disulfide bond of rT1 . Figure 36: Different combinations of disulfide connections and the averaged energies of the 20 best structures generated by CNSsolve 1 .3 accordingly.

Figure 37: SDS-PAGE of the pull-down complex between PPE and biotin-rT1 . The leftmost lane is a protein marker (Bio-rad, US). PPE only lane: purified HNE only. Biotin lane: purified PPE incubated with biotin and NeutrAvidin resin (control). Biotin-rT1 lane: purified PPE incubated with biotin-rT1 and NeutrAvidin resin.

Figure 38: Silver-stained SDS-PAGE of the pull-down complex between HNE and biotin-rT1 . Lane M shows a protein marker (Bio-rad, US); Lane 1 shows the HNE-only lane: purified HNE only; Lane 2 is the biotin lane: purified HNE incubated with biotin and NeutrAvidin resin (control) and Lane 3 is the biotin-rT1 lane: purified HNE incubated with biotin-rT1 and NeutrAvidin resin.

DETAILED DESCRIPTION OF THE INVENTION The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes but not limited to." In case of conflict, the present specification, including explanations of terms, will control.

In a first aspect, the invention relates to a polypeptide comprising, consisting essentially of, or consisting of a peptide having the amino acid sequence of formula: (Xaa)m-Cys-(Xaa)n-Cys-(Xaa)p-Cys-Cys-(Xaa)q-Cys-(Xaa)i-Cys-(Xaa)j , wherein Xaa is any amino acid, preferably any naturally occurring amino acid, wherein independently m is 0 or 1 , n is an integer from 6-9 (i.e. 6, 7, 8, or 9), p is an integer from 2-7 (i.e. 2, 3, 4, 5, 6, or 7), q is 3 or 4, i is an integer from 4-13 (i.e. 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 13), and j is an integer from 1 -6 (i.e. 1 , 2, 3, 4, 5, or 6), and wherein the peptide comprises zero, one, two, three, or more intramolecular disulfide bridges.

The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to polymers of at least two amino acids connected by peptide bonds. The polymer may comprise amino acid analogues or modified amino acids, it may be linear or branched, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or artificially; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a labeling moiety. However, in preferred embodiments, these terms relate to polymers of naturally occurring amino acids, as defined below, which may optionally be modified as defined above, but does not comprise non-amino acid moieties in the polymer backbone.

The term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including both the D and L optical isomers, amino acid analogs (for example norleucine is an analog of leucine) and derivatives known in the art. The term "naturally occurring amino acid", as used herein, relates to the 20 naturally occurring L-amino acids, namely Gly, Ala, Val, Leu, lie, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gin, Asp, Asn, His, Lys, Arg, Tyr, and Trp. The term "peptide bond" refers to a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid. Each "-" in the formula represents a peptide bond. Generally, in the context of the present application, the peptides and polypeptides are shown in the N- to C-terminal orientation. The expression such as (Xaa)m, (Xaa)_n, (Xaa)_P, (Xaa)_q, (Xaa)i, and (Xaa)j as used herein refers to an amino acid sequence having a specific length (m, n, p, q, i, or j amino acids, respectively), wherein each Xaa is independently any amino acid.

In preferred embodiments, m is 0 or 1 and/or n is 6, 8, or 9 and/or p is 2, 5, 6, or 7 and/or q is 3 or 4 and/or i is 4, 9, 1 1 , 12, or 13 and/or j is 1 , 2, or 6.

In certain embodimetns, Xaa in the formula is any one of the above naturally occurring L-amino acid other than Cys. In certain embodiments, at least one of the Xaa in the formula is not Cys. In certain embodiments, in unit (Xaa)_m Xaa is Ala or not present.

In certain embodimetns, in unit (Xaa)_n the second Xaa is Pro and/or the fourth Xaa is Gly and/or the six Xaa is lie. In certain embodiments, in unit (Xaa)q the first Xaa is Asn.

In certain embodiments, in unit (Xaa)i the first Xaa is lie and/or the second Xaa is Phe.

It should be noted that the formula described herein only defines the primary structure of the peptide, i.e. the sequence of amino acids, without including any information about the disulfide bridges (-S-S-) that may be existing in the peptide.

Disulfide bridges are produced by the oxidative folding of two different thiol groups (-SH) present in the peptide. The peptide described herein contains at least six different thiol groups (i.e. six cysteine residues each containing a thiol group) and therefore may form zero, one, two, three, or more intramolecular disulfide bridges.

The number of possible disulfide bridge connectivity patterns (cysteine pairings) increases rapidly with the number of bound cysteines. In order to determine the number and position of the intracellular disulfide bridges of the polypeptide described herein, NMR spectroscopy or other technologies known in the art may be used.

In various embodiments, the polypeptide comprises or consists of

(i) a peptide having the amino acid sequence set forth in any one of SEQ ID NOs:1 -8, preferably SEQ ID NO:1 ; or

(ii) a peptide having the amino acid sequence sharing at least 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, or 74%, preferably at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, or 84%, even more preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, or 94%, most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with the peptide of (i) over its entire sequence, wherein the six cysteine residues in the formula of claim 1 are invariable. The peptides having the amino acid sequences set forth in SEQ ID NOs:1 -8 refer to the following:

The term "sequence identity" as used herein refers to the extent that two sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for peptides) over a window of comparison. This is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base or amino acid residue 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

It should be noted that the sequence identity is always calculated in relation to a reference sequence over its entire length. In this connection, the term "window of comparison", as used herein, refers to a conceptual segment of contiguous nucleotide or amino acid positions wherein a nucleotide or amino acid sequence may be compared to a reference sequence and wherein the portion of the nucleotide or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) while the reference sequence does not comprise additions or deletions for optimal alignment of the two sequences. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 1 981 , 2:482; by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 1970, 48:443; by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. U.S.A. 1988, 85:2444; or by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View, Calif. ; and GAP, BESTFIT, BLAST, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.). The CLUSTAL program is well described by Higgins and Sharp, Gene 1988, 73:237-244; Higgins and Sharp, CABIOS 1989, 5:1 51 -153; Corpet et al., Nucleic Acids Res. 1988, 16:10881 -10890; Huang et al, Computer Applications in the Biosciences 1992, 8:155-1 65; and Pearson et al., Methods in Molecular Biology 1994, 24:307- 331 . Alignment is also often performed by inspection and manual alignment. It is envisaged that the polypeptide may comprise or consist of a plurality of the peptides described herein, each of which independently has the amino acid sequence described above. For example, the polypeptide may comprise or consists of tandem repeats of one or more peptides of SEQ ID NOs:1 -8, more preferably tandem repeats of the peptide of SEQ ID NO:1 . Such polypeptides may be generated on the basis of rT1 -rT8 using peptide grafting strategies known in the art. For example, an amino acid may be substituted by a conservative amino acid, i.e. one having similar structural and/or chemical properties. Conservative amino acid substitutions may be made on the basis of similarity in one or more of the following : polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tyrosine, tryptophan, cysteine and methionine; polar uncharged amino acids include glycine, serine, threonine, asparagine, and glutamine; polar positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In various embodiments, the polypeptide comprises three disulfide bridges that form a cystine- knot structural motif. The term "cystine-knot motif" as used herein refers to a structural motif containing three disulfide bridges, wherein the sections of polypeptide that occur between two of them form a loop through which a third disulfide bond passes, forming a rotaxane substructure. Said structural motif confers exceptional stability to the polypeptide.

In various embodiments, the polypeptide is 22 to 1000, or more preferably 22 to 500, 22 to 200, 22 to 150, 22 to 100, 22 to 75, or 22 to 46 amino acids in length. In preferred embodiments, the polypeptide has a length of at least 22, preferably up to 46, most preferably between 27-39 amino acids. In various embodiments, the polypeptide has a molecular weight of at least 2, preferably up to 8, most preferably between 2-5 kDa.

In various embodiments, the polypeptide or peptide described herein is non-naturally occurring. For example, it may have been artificially modified or contain a sequence not present in nature. It may also be in a reduced state and therefore contain no intramolecular disulfide bridge, or contain one or more intramolecular disulfide bridges that differ from the naturally occurring ones in number and/or position.

Without wishing to be bound to any particular theory, it is believed that the polypeptides described herein, particularly the polypeptide having the amino acid sequence set forth in SEQ ID NO:1 (i.e. roseltide rT1 ), are resistant to acid, proteolytic, and serum-mediated degradation, and are not cytotoxic or hemolytic. It is believed that the polypeptides are capable of inhibiting the activity of neutrophil elastase, preferably human neutrophil elastase. It is further believed that the polypeptides are bioenergetics peptides, meaning that they can penetrate cells, target mitochondria, interact with the delta-subunit of F-type ATP synthase, and increase cellular ATP levels. In this context, techniques are available in the art to allow ascertaining whether a specific polypeptide described herein has the desired properties without undue burden.

In a second aspect, the invention relates to a conjugate comprising or consisting of the polypeptide described herein and at least one agent of interest, which may be, for example, a protein, nucleic acid molecule, compound, small molecule, macromolecule, organic compound, inorganic compound, affinity tags, nanocrystals, or any other molecule with the desired properties suited for the practice of the present invention. The at least one agent of interest may be linked to the polypeptide covalently, non-covalently, or by both covalent and non-covalent interactions.

In various embodiments, the at least one agent of interest is a therapeutic agent, a diagnostic agent, or a targeting agent.

The term "therapeutic agent" as used herein refers to any agent that alleviates a symptom of or prevents development of a disease, disorder, or condition. Preferably, the therapeutic agent may be an agent capable of modulating mitochondrial function or bioenergetics, including but not limited to doxorubicin, lipoic acid, tocopherol, and geldanamycin. The therapeutic agent may also be a chemotherapeutic agent, i.e. any agent used to treat cancer. Chemotherapeutic agents have many mechanisms of action, some of which are non-specific, affecting all cells in the body, while others are specific or targeted to cancer cells. The term chemotherapeutic agent includes all antineoplastic drugs, including small molecules, biologies, immunologic agents, targeted therapies, cytotoxic or cytolytic agents, alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, or any other agent that is used to kill cancer cells, slow or stop cancer cell division, slow or stop cancer cell metastasis or otherwise treat cancer. The term "diagnostic agent" as used herein refers to any agent that can produce a diagnostic signal detectable by any means in a subject. Preferably, the diagnostic agent is an imaging agent, i.e. any agent known to one of skill in the art to be useful for imaging a cell, tissue or a biofilm. More preferably, the diagnostic agent is a medical imaging agent. Examples of medical imaging agent include, but are not limited to, magnetic resonance imaging (MRI) agents, nuclear magnetic resonance imaging (NMR) agents, positron emission tomography (PET) agents, x-ray agents, optical agents, ultrasound agents and neutron capture therapy agents.

The term "targeting agent" as used herein refers to any agent that directs the polypeptide to a relevant site or to a particular cell or cell type in vivo or in vitro. Preferred targeting agents include antibodies and receptor ligands.

The at least one agent of interest may be conjugated to the N and/or C-terminus or a nonterminal amino acid of the polypeptide. In preferred embodiments, however, the agent of interest is conjugated to the N-terminus of the polypeptide using, for example, amine-reactive crosslinker chemistry.

The at least one agent of interest may be linked to the polypeptide by any known method, such as, but not limited to, chemical cross-linking, avidin bridge, glutation-S-transferase bridge, and peptide-cargo fusion proteins. For example, the polypeptide may be biotinylated and the agent of interest may be avidin labelled.

In various embodiments, the conjugate further comprises a linker connecting the at least one agent of interest to the polypeptide. The linker may be cleavable or non-cleavable in nature. In certain embodiments, the linker may comprise a reducible linkage (including but not limited to disulfide), an acid-labile linkage (including but not limited to acetal, ester, and hydrazone), or a protease-labile linkage (including but not limited to cathepsin and furin cleavage site). The attachment point of the linker on the polypeptide can be any part of the polypeptide, preferably in the N-terminus of the polypeptide.

Salts of the polypeptides or conjugates described herein are also within the scope of the invention. Of these salts, preferred are pharmaceutically acceptable salts, including but not limited to salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt etc.), alkaline earth metal salts, ammonium salts, and the like or salts with organic bases, such as dieth- anolamine salts, cyclohexylamine salts and the like.

The polypeptide of the invention may be prepared synthetically, preferably using a commercially available peptide synthesizer. Methods of synthetic peptide synthesis include, but are not limited to liquid-phase peptide synthesis and solid-phase peptide synthesis. Methods to produce peptides synthetically and according protocols are well-known in the art (Nilsson, B L et al. (2005) Annu Rev BiophysBiomolStruct, 34, 91 ). The polypeptide may be prepared recombinantly or produced by in vitro transcription/translation. The polypeptide, if naturally occurring, may also be prepared from natural sources. For example, roseltide rT1 may be isolated and purified from Hibiscus sabdariffa.

The conjugate of the invention may be prepared by further modifying the synthesized peptides or may be prepared altogether synthetically or recombinantly, using standard techniques known to those skilled in the art.

The present invention provides the above peptides, polypeptides, and conjugates preferably in recombinant, synthetic, isolated, or purified form. Without wishing to be bound to any particular theory, it is believed that the polypeptide described herein can enhance the cellular internalization and mitochondria-targeting capability of the agent of interest conjugated thereto. It is also believed that the polypeptide described herein does not substantially affect or hinder the biological activity of the agent of interest conjugate thereto, meaning that the biological activity of the agent of interest as comprised in the conjugate is substantially the same as of the isolated agent of interest in the absence of the polypeptide. It is further believed that there is usually no need, for the agent of interest to become active, to cleave off the polypeptide as soon as the conjugate has been internalized into the target cell or tissue or has reached or crossed the biological barrier towards its target. In a third aspect, the invention relates to a method of inhibiting neutrophil elastase, the method comprising contacting said elastase with the polypeptide or conjugate described herein.

This method may be carried in vitro, ex vivo, or in vivo. In a fourth aspect, the invention relates to a method of delivering an agent of interest to a mitochondrion of a target cell, the method comprising the steps of: (a) providing a conjugate described herein, wherein the agent of interest is conjugated to the polypeptide of the invention ; and

(b) contacting the target cell with the conjugate or a composition comprising said conjugate.

Subcellular targeting of drugs can improve drug efficiency and specificity while minimizing off- target side effects. Mitochondria are important organelles as drug targets for the treatment of cancers, degenerative diseases, cardiovascular diseases, diabetes, and aging. In this context, the present invention provides mitochondria-targeting peptides for use as efficient peptide- based delivery vectors with selective mitochondrial localization. Poor metabolic stability, low cell penetrating properties, and toxicity, amongst others, are the common pitfalls for peptide-based delivery vectors. Unlike other peptides and proteins, the tertiary structures of the polypeptides of the invention are stabilized by multiple intramolecular disulfide bridges in a cystine-knot arrangement. These features make the polypeptides stable against harsh conditions e.g. acid, proteolytic and serum-mediated degradation.

The polypeptides, roseltide rT1 in particular, are efficient cell-penetrating peptides targeting mitochondria with exceptional stability and minimal toxicity. Different types of agents of interest as cargos can be easily conjugated to the polypeptides as detailed above. Therefore, the polypeptides described herein are a novel delivery vector capable of directing exogenous molecules to mitochondria and represent an important tool with great potentials as next- generation targeted drug therapy. For example, roseltide-rT1 -drug conjugates can be encapsulated in cell-type specific drug carriers to assist cell type-specific and organelle-specific drug delivery. In a fifth aspect, the invention relates to a method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to said subject a therapeutically effective amount of the polypeptide or conjugate described herein, or a composition comprising said peptide or conjugate. Alternatively, the invention also covers the use of said peptide or conjugate for the manufacture of a medicament for the treatment or prevention of a disease, disorder, or condition.

The term "subject" is used interchangeably with "individual" and "patient" herein and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

The disease, disorder, or condition may be any disease, disorder, or condition treatable by a polypeptide or conjugated described herein, e.g. cancers, degenerative diseases, cardiovascular diseases, diabetes, and aging.

In certain preferred embodiments, the disease, disorder, or condition is associated with neutrophil elastase such as, without limitation, airway inflammatory diseases selected from the group consisting of particularly cystic fibrosis, asthma, COPD, and pulmonary emphysema.

In certain preferred embodiments, the disease, disorder, or condition is associated with altered (e.g. enhanced or suppressed) mitochondrial function, such as Bioenergetic aging, free radical mediated oxidative injury, Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, dystonia, schizophrenia, non-insulin dependent diabetes mellitus, mitochondrial encephalopathy, lactic acidosis, and stroke, myoclonic epilepsy ragged red fiber syndrome, or Leber's hereditary optic neuropathy.

In even more preferred embodiments, the disease, disorder, or condition is bioenergetic aging, i.e. a constant decline in the body's ability to generate the energy which leads to fatigue and fragility. The polypeptides or conjugates of the invention can counteract bioenergetic aging by, for example, interacting with F-type ATP synthase which in turn increases cellular ATP levels, resulting in energy renewal. Therefore, the polypeptides or conjugates of the invention can be used as adaptogenic nutraceuticals or pharmaceuticals for sustaining general well-being and for the management of bioenergetic aging and the prevention of old age-related diseases such as muscle weakness, heart disease, and degenerative brain diseases.

In certain preferred embodiments, the disease, disorder, or condition is a cancer. The cancers eligible for the treatment by a polypeptide or conjugate of the present invention should be determined on a case-by-case basis, but may include, without limitation, breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renal cancer and cervix cancer. The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. By the terms "effective amount" and "therapeutically effective amount" of a peptide or conjugation of the invention is meant a nontoxic but sufficient amount of said peptide or conjugate to provide the desired effect. A polypeptide or conjugate of the present invention may be administered in the form of a salt, ester, amide, prodrug, active metabolite, analog, or the like, provided that the salt, ester, amide, prodrug, active metabolite or analog is pharmaceutically acceptable and pharmacologically active in the present context. Salts, esters, amides, prodrugs, active metabolites, analogs, and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley- Interscience, 1 992). "Pharmacologically active" (or simply "active") as in a "pharmacologically active" derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

Prior to being used in the treatment in vivo, pharmaceutical formulations composed of the polypeptide or conjugate in association with a pharmaceutically acceptable carrier may need to be formulated. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations.

Depending on the intended mode of administration, the pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, caplets, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature, e.g., in Remington: The Science and Practice of Pharmacy, cited above.

The polypeptide or conjugate of the present invention may be administered orally, parenterally, rectally, vaginally, buccally, sublingually, nasally, by inhalation, topically, transdermal^, or via an implanted reservoir in dosage forms containing conventional non-toxic pharmaceutically acceptable carriers and excipients. The term "parenteral" as used herein is intended to include subcutaneous, intravenous, and intramuscular injection. The amount of the polypeptide or conjugate administered will, of course, be dependent on the particular active agent, the condition or disorder being treated, the severity of the condition or disorder, the subject's weight, the mode of administration and other pertinent factors known to the prescribing physician. In a six aspect, the invention relates to the use of the polypeptide or conjugate described herein as an inhibitor of neutrophil elastase, preferably human neutrophil elastase (SEQ ID NO:9). In a seventh aspect, the invention relates to the use of the polypeptide or conjugate described herein as a mitochondria-selective targeting agent.

In a final aspect, the invention relates to the use of the polypeptide or conjugate described herein as a medicament.

EXAMPLES

Materials and methods Materials

All chemicals and solvents, unless otherwise mentioned, were purchased from Sigma-Aldrich, USA and ThermoFisher Scientific, USA. pGlosensor-20F vector was purchased from Promega, SG. pCMV6-XL5 encoding PAR2 receptor (NM 005242.3) was purchased from Origene, USA. Anti-PAR2 antibody (SAM1 1 ) Alexa Fluor 647 was purchased from Santa Cruz Biotechnology, USA.

Plant materials

Hibiscus sabdariffa were collected from the Nanyang Community Herb Garden, Nanyang Technological University, Singapore (courtesy of Mr. Ng Kim Chuan). The authenticity of samples was determined taxonomically by Lee, S. and Lam, H.J. of the Singapore Botanic Gardens and voucher specimens were deposited at the Singapore Herbarium in Singapore Botanic Gardens (code number: SING 201 5-144). Dried calyces of Hibiscus sabdariffa were purchased from Hung Soon Medical Trading Pte Ltd, Singapore. Screening and profiling

Fresh plant parts of Hibiscus sabdariffa were extracted with water for 1 5 min at room temperature in 1 :1 0 ratio. The aqueous extract was vortexed vigorously and centrifuged at 16,000 x g for 5 min at 4 °C and subjected to flash chromatography by C1 8 solid phase extraction (SPE) columns (Waters, USA). The fractions were eluted with 60% ethanol/0.01 % trifluoroacetic acid (TFA) and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (AB SCI EX 4700 MALDI-TOF/TOF).

Scale-up extraction and purification of Roseltide rT1

Dried calyces (1 kg) of Hibiscus sabdariffa were extracted for 1 5 min with water and centrifuged at 9,000 rpm for 1 0 min at 4 °C (Beckman Coulter, USA) and the supernatant was filtered through 1 μΜ pore size glass fiber filter paper (Sartorius, Singapore). The filtrate was then loaded onto a C18 flash column (Grace Davison, US) and eluted with 60% ethanol/0.01 % TFA. The eluted fractions were then loaded onto an SP Sepharose resin column (G E Healthcare, UK), eluted with 1 M NaCI (pH 3.0), and followed by ultrafiltration (ViVaflow 200, 2000 MWCO hydrostat) (Figure 29A). Further purification was performed by reversed-phase high performance liquid chromatography (RP-HPLC) (Shimadzu, Japan). A linear gradient of mobile phase A (0.05% TFA/H2O) and mobile phase B (0.05% TFA/ACN) was used on the C1 8 column (250 22 mm, 5 μιτι, 300 A) (Grace Davison, US). MALDI-TOF MS was used to identify the presence of roseltide rT1 in the eluted fractions. The average yield of purified roseltide rT1 is 8 mg/kg dried plant material (Figure 29B,C). The eluted fractions were lyophilized for storage at room temperature. S- reduction and S-alkylation

Purified roseltide rT1 (1 mg/mL) was S-reduced by 10 mM dithiothreitol (DTT) in ammonium bicarbonate buffer (25 mM) pH 8 at 37 °C for 30 min, followed by S-alkylation with 60 mM of iodoacetamide (IAM) at 37 °C for 45 min. MALDI-TOF MS was used to confirm the mass shift after S-reduction and S-alkylation.

De novo peptide sequencing

S-alkylated roseltide rT1 (1 mg/mL) were digested with trypsin or chymotrypsin in 5:1 (v/v) ratio in ammonium bicarbonate buffer (25 mM), pH 8 at 37 °C for 10 min. The digested peptide fragments were then analyzed by MALDI-TOF MS followed by MS/MS (AB SCIEX 4700 MALDI-TOF/TOF). De novo peptide sequencing was performed using the ό-ions and y- ions.

Total RNA isolation and next generation transcriptome sequencing

RNA isolation from fresh Hibiscus sabdariffa calyces was performed based on the protocol by Djami-Tchatchou and Straker using CTAB extraction buffer (2% cetyltrimethylammonium bromide, 2% polyvinylpyrrolidone, 1 00 mM Tris-HCI (pH 8.0), 2 mM EDTA, 2 M NaCI, 2% 2- mercapthoethanol) (Djami-Tchatchou & Straker. The isolation of high quality RNA from the fruit of avocado. S. Afr. J. Bot. 78, 44-46 (201 1 )). RNA library construction was performed using 1 μg of total RNA (RIN value >7.0) by lllumina TruSeq mRNA Sample Prep kit (lllumina, Inc. , San Diego, CA, USA). Briefly, poly-A containing mRNA molecules were purified using poly-T-attached magnetic beads. Following purification, mRNA fragmentation was performed using divalent cations under elevated temperature. RNA fragments were reverse-transcribed into first strand cDNA using Superscript II reverse transcriptase (Invitrogen) and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments were subjected to end repair process, the addition of a single 'A' base, and ligation of the indexing adapters. The products were then purified and enriched using PCR to create the final cDNA library. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for lllumina Sequencing platforms) and qualified using the TapeStation D1 000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Indexed libraries were sequenced using the HiSeq2500 platform (lllumina, San Diego, USA) by Macrogen Inc. (Korea). Peptide mapping using a peptidomic approach method

Identification of putative roseltide sequences and confirmation of sequence of roseltide rT1 were performed using methods described by Serra et al (Serra, et al. A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides. Sci. Rep. 6 (201 6)). One-pot reduction and alkylation were performed on the fractionated peptides. The peptide fractions were subjected to 30 mM dithiothreitol (DTT) and 60 mM bromoethylamine (BrEA) in 0.2 M Tris-HCI buffer (pH 8.6) at 55 °C for 60 min and the reaction was quenched using HCI. The reduced alkylated peptide samples were desalted using a C1 8 Sep-pack column (Waters, USA) and dried using SpeedVac (without heating). After re-dissolving the peptide solutions in H20, they were analyzed using LC-MS/MS performed using an Orbitrap Elite mass spectrometer (Thermo Scientific Inc., Bremen, Germany) coupled with a Dionex UltiMate 3000 UHPLC system (Thermo Scientific Inc., Bremen, Germany). Samples were sprayed using a Michrom's Thermo CaptiveSpray nanoelectrospray ion source (Bruker-Michrom Inc, Auburn, USA) and separation was perfored using a reverse phase Acclaim PepMap RSL column (75 μιτι ID χ 1 5 cm, 2 μιτι particles; Thermo Scientific). The mobile phase was 0.1 % formic acid (FA) as eluent A and 90% ACN 0.1 % FA as eluent B, with a flow rate of 0.3 μί/ιτιίη. A 60 min gradient was used for the elution as follows: 3% B for 1 min, 3-35% B over 47 min, 35-50% B over 4 min, 50- 80% B over 6 s, 80% for 78 s; then, it was reverted to the initial state over 6 s and maintained for 6.5 min.

The Thermo Scientific Orbitrap Elite mass spectrometer was set to positive ion mode using LTQ Tune Plus software (Thermo Scientific Inc. , Bremen, Germany) for data acquisition, alternating between a Full FT-MS (350-3000 m/z, resolution 60.000, with 1 μεοΒη per spectrum) and a FT-MS/MS scan applying 65, 80, and 95 ms ETD activation times (150— 2000 m/z, resolution 30.000, with 2 μεοΒη averaged per MS/MS spectrum). The 3 most intense precursors with a charge >2+ were isolated with a 2 Da mass isolation window and fragmented. The automatic gain control (AGC) for Full MS and MS was set to 1 χ 1 0⁶ and the reagent AGC was 5 χ 1 0⁵. Data analysis was performed using PEAKS studio (version 7.0, Bioinformatics Solutions, Waterloo, Canada), where 1 0 ppm MS and 0.05 Da MS/MS tolerances were applied. A false discovery rate of 0.1 % was applied to accept the sequences.

Acid and proteolytic stability of roseltide rT1 Acid stability

0.1 M of purified roseltide rT1 was dissolved in 0.2 M HCI or phosphate buffered saline (PBS) (control) and incubated at 37 °C. Samples were collected at various time points (0, 1 5, 30, 45, 60 and 120 min). Enzymatic stability

0.1 M of purified roseltide rT1 was dissolved in 1 00 mM Tris buffer (pH 8) and incubated with trypsin (Catalog number: T1426, Roche Applied Science, US) in a 50:1 (w/v) ratio at 37 °C. 0.1 M of purified roseltide rT1 was dissolved in 0.2 M HCI and incubated with pepsin (Catalog number: 031 1 7901 001 , Roche Applied Science, US) in a 50:1 (w/v) ratio at 37 °C. Trypsin substrate and DALK (KRPPGFSPL-NH2) were used as controls, respectively. Samples were collected at various time points (0, 15, 30, 45, 60 and 120 min).

Human serum stability

0.1 M of purified roseltide rT1 was prepared in 25% human serum in DMEM medium without phenol red. The test samples were incubated at 37 °C. DALK was used as a positive control. Samples were collected at various time points (0, 24 and 48 h). The collected samples were subjected to protein precipitation with 1 00% ethanol and centrifugation at 1 80,000 g for 5 min at 4 °C. The supernatant was collected for analysis. Analysis for stability assays

All collected samples from various stability assays were analyzed by RP-UHPLC with a linear gradient of mobile phase A (0.05% TFA/H2O) and mobile phase B (0.05% TFA/ACN) on C1 8 column (3.6 μιτι particle size; 2.1 mm internal diameter; 10 cm length) (Phenomenex, US). The resulting peaks were collected and the identified by MALDI-TOF MS. The results were expressed as percentage of initial concentration using the peak area of the UHPLC profile.

Human neutrophil elastase inhibition assay

Human neutrophil elastase (HNE) activity was determined by measuring the release of p- nitroanilide at 405 nm at 37 °C using N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide as substrates. Purified roseltide rT1 was incubated with 1 .75 U/mL HNE and 0.6 mM substrate in 1 00 mM Tris buffer (pH 8.0) at 37 °C; absorbance was measured continuously for 1 h. A synthetic elastase inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, was used as a positive control. The results were presented as normalized initial velocity.

Peptide biotinylation and pull-down assay Purified roseltide rT1 was biotinylated with EZ-Link NHS-LC-biotin (Thermo Fisher Scientific, US) in 100 mM phosphate buffer at a pH of 7.8. Biotinylation was carried out at room temperature for 2 h and the biotinylated peptide was then identified and purified by MALDI- TOF MS and RP-HPLC. Pull-down assay was performed using NeutrAvidin UltraLink Resin (Thermo Fisher Scientific, US). Briefly, the resin was washed with PBS three times and incubated with biotinylated roseltide rT1 or biotin (control) at room temperature with rotation for 1 h. The resin was washed again with excess PBS six times before incubation with HNE overnight at 4 °C with rotation. The resin was washed with excess PBS for six times and dissociation was performed by the addition of 6x loading dye with 2-mercaptoethanol and was heated at 85 °C for 1 0 min. The samples were then resolved in 12% SDS-PAGE for 1 .5 h. Silver staining was performed for protein visualization.

Cell culture

Huh7 (human liver carcinoma cells), A549 (human lung adenocarcinoma epithelial cells) cells, and Chinese Hamster Ovary-K1 cells (CHO-K1 ) were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12 containing 15 mM HEPES and L-glutamine and supplemented with 1 0% fetal bovine serum, 1 00 U/ml_ of penicillin, and streptomycin. The cells were grown in a 5% CO2 humidified incubator at 37 °C. CHO-K1 cells were transfected with pGlosensor-20F plasmid by electroporation and selected using 500 μg/mL hygromycin. CHO-K1 cells stably expressing the pGlosensor-20F construct (cAMP-CHO) were then transfected with pCMV6-XL5 encoding PAR2 receptor (NM 005242.3) and selected using 500 μg/mL G41 8. Stable cell lines co-expressing both pGlosensor-20F and PAR2 receptor constructs (PAR2-cAMP-CHO) were maintained with 500 μg/mL hygromycin and 500 μg/mL G41 8.

Flow cytometry analysis

PAR2-cAMP-CHO and cAMP-CHO cells were harvested and collected by centrifugation at 500 g for 5 min at 4 °C. The cell pellet was stained with the anti-PAR2 antibody (SAM1 1 ) Alexa Fluor 647 (1 :1 00, Santa Cruz Biotechnology, USA) in serum-containing medium for 30 min on ice. The pellet was then washed three times and samples were analyzed by flow cytometry. 10,000 cells were analyzed using the BD LSRFortessa X-20 flow cytometer.

Immunofluorescence analysis

PAR2-cAMP-CHO and cAMP-CHO cells were seeded on an 8-well chamber slide (ibidi, Germany). The slides were washed gently with PBS, fixed in 4% paraformaldehyde for 10 min, and permeabilized in PBS containing 0.25% triton X-1 00 for 15 min. The slides were blocked in PBS containing 3% BSA for 1 h and then incubated with anti-PAR2 antibody (SAM1 1 ) Alexa Fluor 647 (1 :50, Santa Cruz Biotechnology, USA) for 1 h. After PBS washing, the slides were mounted with Fluoroshield containing DAPI (Sigma, USA) and observed under a Zeiss LSM 71 0 confocal microscope (ZEISS, Germany). Glosensor cAMP assay

Glosensor cAMP assays were performed according to the manufacturer's instructions. Briefly, PAR2-cAMP-CHO cells were grown until they reached confluency in a white-walled, clear- bottom 96 well plate. The culture medium was replaced with 6% (v/v) Glosensor cAMP substrate in C02-independent medium in the dark at room temperature for 1 h. Luminescent intensity was measured using a microplate reader (Tecan Infinite 200 Pro, Switzerland) ; the plate was pre-read for 30 s to establish basal luminescent level. Following treatment, luminescent levels were continuously monitored for 20 min. The results are presented as fold change relative to basal luminescent levels and were quantified using the area under the curve.

Cell viability assay

Cell viability was measured using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay. Briefly, cells were treated with roseltide rT1 or 0.1 % triton X-100 (positive control) for 24 h. MTT (final concentration 0.5 mg/mL) was added and incubated for 3 h at 37 °C. Dimethyl sulfoxide was then added to dissolve the insoluble formazan crystal. The absorbance was measured at 550 nm using a microplate reader (Tecan Infinite 200 Pro, Switzerland).

NMR spectroscopy and structure determination of roseltide rT1

A sample of roseltide rT1 for NMR spectroscopy was prepared by dissolving the lyophilized peptide in water containing 5% D2O at a final peptide concentration of 1 .5 mM. For H/D exchange NMR experiment, the sample was dissolved in solution with 1 00% D2O immediately before the experiment. All NMR spectra were collected at a sample temperature of 298 K on a Bruker AVANCE II 600 MHz NMR spectrometer equipped with four RF channels and a 5 mm z-gradient TCI cryoprobe. Phase-sensitive two-dimensional ¹ H, ¹ H- TOCSY and NOESY spectra were recorded with a spectral width of 12 ppm. For water suppression, excitation sculpting with gradients was applied to all NMR experiments. TOCSY and NOESY spectra were obtained with mixing times of 80 ms and 200 ms respectively. The proton chemical shifts were referenced to external sodium 2,2-dimethyl-2-silapentane-5- sulfonate (DSS). All measurements were recorded with 2048 complex data points and zero- filled to 2048 x 512 data matrices. Time domain data in both dimensions were multiplied by a 90 °-shifted squared sine bell window function prior to Fourier transformation. Baseline correction was applied with a fifth order polynomial. NMR data were acquired and processed by TopSpin (Bruker BioSpin). The NMR spectra were processed with NMRpipe. Sequence- specific assignments were achieved with 2D TOCSY and NOESY and NOEs were performed using SPARKY. Distance restraints were derived based on the intensities of NOE cross peaks, which were divided to three classes: strong, 1 < d < 1 .8; medium, 1 .8 < d < 3.4; weak, 3.4 < d < 5. Hydrogen bond restraints were determined based on H/D exchange 1 D NMR experiment, in which amide protons exchanged with solvent deuterium for 24 hours in 298 K. The hydrogen bond restraints were defined as: N-O, 0.8-3.3 A; HN-O, 0.6-2.2 A. Dihedral angle restraints were derived from the ³JHN-H_q coupling constant measured in 1 D ¹ H NMR spectrum. The backbone Φ angle was considered between -1 00 ° to -1 60 ° if the coupling constant was larger than 8 Hz. Three-dimensional structures were reconstructed using CNSsolve 1 .3. The 6 cysteines were assumed to form disulfide bonds in structure calculation. Structures were displayed with Chimera and Pymol and validated with the online server PDBsum. In silico modeling

The in silico docking was performed using automatic protein-protein docking server ClusPro Version 2.0. Both the NMR structure of roseltide rT1 and the crystal structure of HNE (PDB entry:^"! HNE) were uploaded to the server. It uses rigid body docking protocol and the model was generated based on electrostatic potentials.

Statistical analyses

Statistical comparisons were performed using GraphPad Version 6.0d (USA). Data were analyzed using one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc tests. Data were expressed as mean ± S.E.M and P < 0.05 was considered to be statistically significant.

Example 1: Identification and Characterization of Roseltide rT1

Isolation of roseltide rT1 from Hibiscus sabdariffa:

Mass spectrometry profiling of the aqueous extracts of Hibiscus sabdariffa revealed the presence of a cluster of strong signals in the mass range of 2-4 kDa in the calyces, capsules and flowers (Figure 1 ). The inventors focused on one of the strongest signals of this cluster, the 2620 Da-peak which was found in the calyces, capsules and flower extracts. The 2620 Da-peak was shown to be a CRP with six cysteine residues based on a mass increase of 348 Da after S- reduction by dithiothreitol and S-alkylation by iodoacetamide (Figure 28). To characterize this CRP, a scale-up aqueous extraction was carried out using the calyces of Hibiscus sabdariffa. The crude aqueous extract was fractionated by C18 reversed-phase and strong cation- exchange flash chromatography, followed by ultrafiltration using a membrane with molecular weight cut-off of 2000 Da. The CRP-enriched fraction was further purified by RP-HPLC (Figure 29), and the 2620-Da CRP was designated roseltide rT1 . 6-8 mg of purified roseltide rT1 was obtained per Kg of dried calyces. To determine the amino acid sequence of roseltide rT1 , the purified roseltide rT1 was fully S- reduced and S-alkylated followed by digestion with either trypsin or chymotrypsin. The digested peptide fragments were analyzed by MALDI-TOF MS, followed by MS/MS sequencing. Analysis using the ό-ions and y-ions revealed that roseltide rT1 is a 27-residue peptide with six cysteine residues (Figure 2). The amino acid sequence of rT1 was confirmed by transcriptomic analysis. Transcriptome analyzes showed that roseltide rT1 was biosynthesized as a 90-residue precursor with three domains: a 28-residue N-terminal signal peptide, a 35-residue pro-domain and a 27-residue C-terminal mature peptide (Figure 3). Using the asparaginyl endopeptidase cleavage site and the cysteine spacing pattern of roseltide rT1 in its transcriptomic database search, additional seven putative roseltide sequences (rT2-rT8) were identified (Figure 3 & Table 1 ). Using a high-throughput peptidomic method for peptide sequencing developed by the inventors' laboratory (Serra, et al. A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides. Sci. Rep. 6 (201 6)), the inventors identified the presence of rT7 in the aqueous extract of Hibiscus sabdariffa calyces (Figure 30). Table 1 : Putative amino acid sequences of roseltides.

Amino acki sequence Cafcuistesi Expression fevst

MW

-eis&ssi- - -exviw ss-ccKSPCC r¾¾ ic& 2«2S

-ct,ss¾S8— csm&SBssx v-o vvsime 3281

s?3 ^••«eXS8S SM»CS0 CCS -KKC&X¾T)?W xc¾ 32S8 mS!>iA s?4 ~C!iIS6SF~--CYS-VS¾?-CCS-S irSi'SIFi>fTgXH~CV 337$ mRHA. r-rs -CSS3WXV CSfc-CtSfc-CCB^•<MtO'XV8XcS'*J∞∑S" lKS( 34 ?S mRNA ιϊδ A &PXOJkX CKJ ~~«¾S8CS- 3KCS£-XS2S£«i'S«-C∑> - 3<S 92

si? -ssrasei M*- css ccesmc u sr sxf - vcsv - €S£ tnSNA.. pfoteif!

-CKi?V«&S CSZ XSGZ"Cl tl r21 $( z$VXVm «028

NMR structure of roseltide rT1 :

The cysteine spacing of all eight roseltides contains a CC motif which provides a clue to their putative disulfide connectivity as a cystine-knot. Using peptide mapping, NMR, and X-ray crystallography, the inventors have previously demonstrated in cystine-knot a-amylase inhibitors (CKAI) (Figure 4A) that the six-Cys-containing CRPs having a CC motif of 111 and CIV such as roseltides are often arranged in a cystine-knot structural motif (Cys l-IV, Cys ll-V and Cys III- Vl)21 '22.23. To characterize the structural fold of roseltide rT1 , the solution structure of rT1 was determined using 2D ¹ H, ¹ H- TOCSY and NOESY NMR spectra. The sequential assignment was done based on the NOE cross peaks between HN, and Ηα,-ι as well as the other side chain protons of residue i-1 (Figure 31 and 32). When performing the assignment, the inventors compared the NOESY spectrum and the TOCSY spectrum to differentiate the intra-residue and inter-residue NOEs. The amide proton of residue i should have NOE cross peaks with the side chain protons of the residue i-1 . The pattern of the peaks in TOCSY of each amide proton stripe should correspond to its specific residue. Based on these strategies, the sequential assignment was completed. More than 95% of the peaks in the NOESY spectrum were assigned. H/D exchange NMR experiment indicated that the residue C26 and G5, I25 and I22 as well as I2 and C1 6 should be involved in hydrogen bonds, which was consistent with the NOE cross peaks between the amide protons of them accordingly. This observation further confirms the sequential assignment. Twenty structures with the lowest energy among 100 structures were generated by CNSsolve 1 .335 (Figure 33). The 20 structures are highly converged, of which the backbone RMSD and the heavy atom RMSD are 0.71 ± 0.14 A and 1 .19 ± 0.26 A respectively (Table 2). The ensemble of the 20 best structures has been deposited to Protein Data Bank (PDB) with the accession number 5GSF. The chemical shifts have been submitted to Biological Magnetic Resonance Bank (BMRB), of which the accession number is 26874 (Figure 34).

Table 2: Parameters and restrains of structure calculation of roseltide rT1 .

The proton chemical shifts were uploaded to the online server CSI 3.0 (http://csi3.wishartlab.com/cgi-bin/index.php) to predict the secondary structure, indicating that only residue F23-C26 might be edge β strand while the other residues were coil. The structure of rT1 generated by simulated annealing contains no a helix or β strand. The two prolines both adopt trans form, which are supported by the NOE cross peak between Ηδ, and HNi-i of the proline and the previous residue respectively. The disulfide bonds Cys8-Cys21 and Cys15- Cys26 cross in the center of rT1 . The disulfide bond Cys1 -Cys16 makes the N terminus bend (Figure 4B).

Further evidence supporting the Cys1 -Cys16, Cys8-Cys21 and Cys15-Cys26 disulfide linkages were observed from the Ηβ-Ηβ NOE cross peaks (Figure 35). To confirm the disulfide connectivity, all the six cysteines were assumed reduced in the simulated annealing by CNSsolve 1 .335. The 100 structures were highly converged. The backbone RMSD and the heavy atom RMSD of the 20 best structures are 0.73 ± 0.27 A and 1 .25 ± 0.26 A respectively. The averaged energy of the 20 best structures is similar to that of the 20 best structures with the 3 disulfide bonds imposed (Cys1 -Cys16, Cys8-Cys21 and Cys15-Cys26). The structures generated with and without the disulfide bond imposed are very similar, except for the sidechains of the six cysteines (Figure 33). Moreover, another 14 disulfide patterns were assumed in structure calculations respectively. The average energy of the 20 best structures for each combination was compared with the one with the pattern: Cys1 -Cys16, Cys8-Cys21 and Cys15-Cys26. The disulfide pattern of: Cys1 -Cys1 6, Cys8-Cys21 and Cys15-26 has the lowest average energy among the 15 combinations (Figure 36). These results strongly suggest that the disulfide connectivity of roseltide rT1 exists in a cystine-knot structural motif.

Protein tertiary structure comparison was conducted using the SuperPose software Version 1 .043 for the wrightide Wr-AI1 (PDB entry: 2MAU)22 and alstotide As1 (PDB entry: 2MM6)23 which displays similar cystine-knot fold (Figure 4C). The RMSD values between the superimposed structure of wrightide Wr-AI1 and roseltide rT1 were 0.515 A and 1 .036 A for all Ca and heavy atoms, respectively. The RMSD values between the superimposed structures of alstotide As1 and roseltide rT1 were 0.534 A and 1 .003 A for all Ca and heavy atoms, respectively. Based on the electrostatic potential surface of roseltide rT1 , a negatively-charged region was observed. This is created by the side-chain of Arg4 residue which is positioned outwards.

Acid and proteolytic stability of roseltide rT1 :

Intact CRPs are highly cross-linked by multiple disulfide bridges which confers their high stability. To determine the acid and proteolytic stability of roseltide rT1 , roseltide rT1 was incubated in 0.2 N HCI, or with proteinases (trypsin or pepsin), or in 25% human serum. The results demonstrated that roseltide rT1 was resistant against acid, proteinase and human serum- mediated degradation (Figure 5). Roseltide rT1 is not cytotoxic:

To determine the cytotoxicity of roseltide rT1 , cell viability was measured by MTT assay. Treatment with roseltide rT1 of concentrations up to 1 00 μΜ for 24 h did not affect the viability of Huh7 (human liver carcinoma cells) and A549 (human lung adenocarcinoma epithelial cells) cells (Figure 6).

Example 2: Roseltide rT1 is a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa

Roseltide rT1 inhibited human neutrophil elastase:

Previous studies showed that the extract of Hibiscus sabdariffa calyces contains proteinase inhibitors against elastasel O, however, the active compounds are yet to be reported. To characterize the biological activities of roseltide rT1 , its effects on the enzymatic activities of human neutrophil elastase was examined. As shown in Figure 7A, roseltide rT1 inhibited the enzymatic activities of human neutrophil elastase in a dose-dependent manner, with an ICso of 0.47 μΜ. This was comparable to the activity of a synthetic elastase inhibitor (MeOSu-AAPV- CMK). Roseltide rT1 was also screened against trypsin and porcine pancreatic elastase without observable inhibitory effects up to 10 μΜ (data not shown).

Roseltide rT1 showed protein interactions to human neutrophil elastase:

Pull-down assays were performed to determine the binding of roseltide rT1 to human neutrophil elastase. Purified biotin-rT1 was characterized by MALDI-TOF MS and RP-HPLC (Figure 7B). Following biotinylation, the MS profile showed an increase in mass from 2620 Da to 2960 Da. Biotin-rT1 inhibits human neutrophil elastase (Figure 7C) and was able to pull-down human neutrophil elastase with a band of approximately 25 kDa (Figure 7D). Pull-down assay was also performed using porcine pancreatic elastase however, biotin-rT1 did not show protein interactions with porcine pancreatic elastase (Figure 37). Roseltide rT1 inhibited neutrophil elastase-induced cAMP accumulation:

PAR2 is a GPCR responsible for the cellular effects of neutrophil elastase. To demonstrate the cellular effects of neutrophil elastase inhibition in vitro, the effects of roseltide rT1 on neutrophil elastase-stimulated cAMP accumulation were evaluated. In this study, CHO-K1 cells stably transfected with Glosensor cAMP biosensor (cAMP-CHO cells) was used to provide direct and real-time measurement of cAMP accumulation in live cells. The cAMP-CHO cells were further overexpressed with the gene of PAR2 receptor (PAR2-cAMP-CHO) cells. The expression levels of PAR2 were confirmed by flow cytometry and confocal microscopy while the function of cAMP biosensor was assessed using a cAMP activator, forskolin (10 μΜ) (Figure 8A-C). Similar to previous findings, human neutrophil elastase stimulated intracellular cAMP accumulation in PAR2-cAMP-CHO cells. Co-incubation of human neutrophil elastase with roseltide rT1 significantly suppressed cAMP accumulation (Figure 8D).

In addition, in comparison to the knottin-type elastase inhibitors from the squash inhibitors family (MCEI-I to MCEI-IV), there is no sequence homology to roseltide rT1 other than classification as a cystine-knot peptide24^>25 (Table 3).

Table 3: Sequence comparison between Roseltide rT1 and MCEI-I-IV. oseifiite Amino acid sequence Reference

— -cx GGicim&s&c- ~ - SSFGCX GICA This study S2 Cgl»X^JEC RSS»~ CLAQCX CVDGHCG {23}

~ -BmiCgliXiSiEC SDSB-CL QCl— -CVE>GHCG

MCEI-2X (23)

MCKX~X2X CPIilWMECKKSSB- Clc&QCI— -CVIXSKCG {23}

MCEI-IV SSKRXCeLXfcmCKimSC-CXAOei CVBSHCG {24}

Example 3: rT1 is a Mitochondria-targeting and bioenergetics ATP-elevating peptide from Hibiscus sabdariffa As shown in Figure 10, the N-terminal modification of roseltide rT1 using Cy3-NHS ester in 100 mM phosphate buffer, pH 7.8 was carried out. The Cy3-labeled rT1 (Cy3-rT1 ) was then identified and purified by MALDI-TOF MS and RP-HPLC.

As shown in Figures 1 1 -12, the quantitative measurement for the cellular uptake of Cy3-rT1 in WI-38 and HUVEC-CS cells was carried out by flow cytometry. 1 μΜ Cy3-rT1 was incubated in WI-38 and HUVEC-CS cells for up to 1 hour. The mean fluorescence intensity of the cell population was increased and reached plateau in 1 hour for both WI-38 and HUVEC-CS cells. Figure 13 shows the orthogonal view and 3D rendering of the Z-stack images of the live-cell imaging by confocal microscopy after incubation with 1 μΜ Cy3-rT1 for 15 min in HUVEC-CS cells. The confocal images showed that Cy3-rT1 was internalized and distributed throughout the cell, but does not accumulate in the nucleus.

As shown in Figure 14, the cellular uptake of Cy3-rT1 was not affected by serum. As shown in Figures 1 5-17, the cellular uptake of Cy3-rT1 was mainly mediated by endocytosis. Direct cell penetration and endocytosis are the main mechanisms involved in the cellular uptake of cell penetration peptides and cyclotides. Endocytosis is an energy-dependent active transport of molecules. To deduce the cellular uptake mechanisms, Cy3-rT1 1 was incubated with WI-38 (Figure 15) and HUVEC-CS cells (Figure 1 6) at 4 °C for 1 hour. As illustrated in Figure 15-16, the cellular uptake of Cy3-rT1 at 4 °C was significantly reduced compared to at 37 °C. This results indicated that the cellular uptake mechanisms of Cy3-rT1 was largely involving energy- dependent endocytosis. To further substantiate the findings, HUVEC-CS cells were pre- incubated with different endocytosis inhibitors for 30 min before incubation with Cy3-rT1 for 1 h. The results showed that endocytosis inhibitors including dynasore (dynamin-dependent endocytosis inhibitor) inhibited the cellular uptake of Cy3-rT1 , indicating the involvement of endocytosis (Figure 17). As shown in Figure 18, CHO-K1 cells internalized Cy3-rT1 in a time-dependent manner and the mean fluorescence intensity at different time points were significantly higher than in glycosaminoglycan-deficient PgsA-745 cells (P<0.05). Subcellular localization was conducted to investigate the endosomal escape properties of Cy3- rT1 . The inventors used organelle-specific fluorescent trackers to carry out the co-localization experiment with live-cell confocal microscopy. Results illustrated in Figure 19 revealed that Cy3- rT1 co-localized with MitoTracker Green FM (Pearson's correlation : 0.76), signifying that Cy3- rT1 escaped from endosomes and was delivered to the mitochondrial compartment. As a control, roseltide rT7 was also fluorescent-labeled with Cy3-NHS ester (Cy3-rT7). Confocal microscope analysis showed that it does not co-localize with MitoTracker Green FM. To further support the co-localization experiments, the inventors incubated N-terminal biotinylated roseltide rT1 (Biotin-rT1 ) with HUVEC-CS cells for 1 h. The resultant mixture was then subjected to mitochondria isolation experiments. Immunoblot results showed that biotinylated roseltide rT1 was accumulated in the mitochondria (Figure 20). Quality of subcellular fractionation was demonstrated by protein markers: GAPDH (cytoplasm) and CoxlV (mitochondria).

Pull-down assay followed by immunoblotting showed that biotin-rT1 interacts with TOM20, a mitochondria import receptor (Figure 21 ). To substantiate these results in an in vitro model, the inventors established a TOM20-GFP stably-transfected HEK293 cells for co-localization experiments using live-cell confocal microscopy. The results showed that Cy3-rT1 co-localized with TOM20-GFP in vitro (Pearson's correlation: 0.74) (Figure 22). Figure 23 showed the x-ray crystal structure of the cytosolic domain of rat mitochondrial protein input receptor TOM20 (PDB entry: 2V1 T; chain A).

The inventors also performed rigid body docking to model the interaction between roseltide rT1 and TOM20 by ClusPro Version 2.0 server. As illustrated in Figure 23, the predicted interaction surface between roseltide rT1 and TOM20 is located at the hydrophobic groove of TOM20. As shown in Figure 24, roseltide rT1 induced mitochondrial membrane hyperpolarization and increased mitochondria ROS levels in isolated mitochondria suggesting increased mitochondria respiration. Figure 25 shows that roseltide rT1 increased cellular ATP levels.

The inventors performed pull-down assays followed by LC-MS/MS analysis to search for potential binding targets related to mitochondria function. Among the three experimental repeats, a total of 202 proteins can be identified from the roseltide rT1 samples, but not in the control samples. Two proteins were identified in all three experimental repeats. This includes human DnaJ homolog subfamily C member 3 (Gene accession: Q1321 7) and human mitochondrial ATP synthase subunit O (Gene accession: P48047) (Figure 26). Human mitochondrial ATP synthase subunit O, also known as ATP50 or Oligomycin sensitivity conferral protein (OSCP) is the 5-subunit of F- type ATP synthase. Mutations to this subunit have been reported to affect ATP synthesis efficiency. To confirm with the LC-MS/MS results, the inventors performed pull-down assay followed by western blot analyses (Figure 27). The western blot results supported that roseltide rT1 showed protein interactions with ATP50. To substantiate the results, the inventors have performed in silico modeling to gain insights into the interaction between roseltide rT1 and ATP50. The predicted structure of ATP50 was adopted from MODBASE based on the crystal structure of bovine ATP synthase (PDB entry: 2wssS). Using Cluspro V2.0 server, the inventors modeled the interactions between roseltide rT1 and ATP50. As illustrated in Figure 27, it was speculated that the intercysteine loop 1 of roseltide rT1 is important to form ionic interactions with ATP50.

Table 4. Other amino acid sequences described in the present applicaton.

Name/Note Sequence SEQ ID NO

Huamn MTLGRRLACLFLACVLPALLLGGTALASEIVGGRRARPHAW SEQ ID NO:9 neutrophil PFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAVR

elastase VVLGAHNLSRREPTRQVFAVQRIFENGYDPVNLLNDIVILQL

NGSATINANVQVAQLPAQGRRLGNGVQCLAMGWGLLGRr

RGIASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFGDS GSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVNWi:

SIIQRSEDNPCPHPRDPDPASRTH

Peptide of CIPRGGICLVALSGCCNSPGCIFGICA SEQ ID NO:10 Figure 2A, 2D

Peptide of GGICLVALSGCCNSPGCIFGICA SEQ ID NO:1 1 Figure 2C

Peptide of CIPRGGICL SEQ ID NO:12 Figure 2E

Peptide of CIPRGGICLVAL SEQ ID NO:13 Figure 2F

Ac2 of Figure CRPVGTRCDGVINQCCDPYWCTPPIYGWCK SEQ ID NO:14 4

Wr-AI1 of CAQKGEYCSVYLQCCDPYHCTQPVIGGICA SEQ ID NO:15 Figure 4

AS1 of Figure CRPYGYRCDGVINQCCDPYHCTPPLIGICL SEQ ID NO:16 4 AS3 of Figure CVPRFGRCDGIINQCCDPYLCTPPLVGICT SEQ ID NO:17 4

AS4 of Figure CVPQYGVCDGIINQCCDPYYCSPPIYGHCI SEQ ID NO:18 4

Human M AAP AVSG LS RQV RC FSTS VVR P F AKLV R P P VQ V YG 1 EG R SEQ ID NO:19 ATP50 of YATALYSAASKQNKLEQVEKELLRVAQILKEPKVAASVLNP

Figure 27 YVKRSIKVKSLNDITAKERFSPLTTNLINLLAENGRLSNTQG

VVSAFSTMMSVHRGEVPCTVTSASPLEEATLSELKTVLKSF LSQGQVLKLEAKTDPSILGGMIVRIGEKYVDMSVKTKIQKLC RAMREIV

Peptide of LSGCCNSPGCIFGICA SEQ ID NO:20 Figure 30

Peptide of SEQ ID NO:21 Figure 30 SGCCNSPGCIFGICA

Peptide of SEQ ID NO:22 Figure 30 VALSGCCNSPGCIFGICA

Peptide of SEQ ID NO:23 Figure 30 CIPRGGICLVA

Peptide of SEQ ID NO:24 Figure 30 CIPRGGICLVALSGCCNSPGCIFGICA

Peptide of SEQ ID NO:25 Figure 30 SPGCIFGICA

Peptide of SEQ ID NO:26 Figure 30 GCCNSPGCIFGICA

Peptide of SEQ ID NO:27 Figure 30 ALSGCCNSPGCIFGICA

Peptide of SEQ ID NO:28 Figure 30 CVSSGIVDACSECCEPD

Peptide of SEQ ID NO:29 Figure 30 CVSSGIVDACSECCEPDKCIIMLPTWPPRYVCSV

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1 . Polypeptide comprising or consisting of a peptide having the amino acid sequence of formula:

(Xaa)m-Cys-(Xaa)n-Cys-(Xaa)p-Cys-Cys-(Xaa)_q-Cys-(Xaa)i-Cys-(Xaa)j, wherein Xaa is any amino acid, preferably any naturally occurring amino acid, wherein independently m is 0 or 1 , n is an integer from 6-9, p is an integer from 2-7, q is 3 or 4, i is an integer from 4-13, and j is an integer from 1 -6, and wherein the peptide comprises none, one, or more intramolecular disulfide bridges.

2. The polypeptide of claim 1 comprising or consisting of

(i) a peptide having the amino acid sequence set forth in any one of SEQ ID NOs:1 -8; or (ii) a peptide having the amino acid sequence sharing at least 65%, preferably at least 75%, even more preferably at least 85%, most preferably at least 95% sequence identity with the peptide of (i) over its entire sequence, wherein the six cysteine residues in the formula of claim 1 are invariable.

3. The polypeptide of claim 1 or 2, wherein the peptide has the amino acid sequence set forth in SEQ ID NO:1 .

4. The polypeptide of any one of claims 1 -3, wherein the peptide comprises three disulfide bridges that form a cystine-knot structural motif.

5. The polypeptide of any one of 1 -4, wherein the peptide has a length of at least 22, preferably up to 46, most preferably between 27-39 amino acids.

6. The polypeptide of any one of claims 1 -5, wherein the peptide has a molecular weight of at least 2, preferably up to 8, most preferably between 2-5 kDa.

7. Conjugate comprising or consisting of the polypeptide of any one of claims 1 -6 and at least one agent of interest.

8. The conjugate of claim 7, wherein the agent of interest is a therapeutic agent, a diagnostic agent, or a targeting agent.

9. The conjugate of claim 7 or 8, wherein the agent of interest is conjugated to the N-terminus of the peptide.

10. The conjugate of any one of claims 7-10, wherein the conjugate further comprises a linker connecting the agent of interest to the peptide.

1 1 . Method of inhibiting neutrophil elastase, the method comprising contacting said elastase with the polypeptide of any one of claims 1 -6 or the conjugate of any one of claims 7-10.

12. Method of delivering an agent of interest to a mitochondrion of a target cell, the method comprising the steps of:

(a) providing a conjugate of any one of claims 7-10, wherein the agent of interest is conjugated to the polypeptide of any one of claims 1 -6; and

(b) contacting the target cell with the conjugate of any one of claims 7-10 or a composition comprising said conjugate.

13. Method of treating or preventing a disease, disorder, or condition in a subject, the method comprising administering to said subject a therapeutically effective amount of the polypeptide of any one of claims 1 -6, a conjugate of any one of claims 7-10, or a composition comprising said peptide or conjugate.

14. The method of claim 13, wherein the disease, disorder, or condition is associated with neutrophil elastase.

15. The method of claim 14, wherein the disease is an airway inflammatory disease selected from the group consisting of cystic fibrosis, asthma, COPD, and pulmonary emphysema.

16. The method of claim 13, wherein the disease, disorder, or condition is associated with altered mitochondrial function.

17. The method of claim 16, wherein the disease, disorder, or condition is bioenergetic aging.

18. The method of claim 13, wherein the disease, disorder, or condition is a cancer.

19. The method of any one of claims 13-17, wherein the subject is a mammal, preferentially a human.

20. Use of the polypeptide of any one of claims 1 -6 and conjugate of any one of claims 7-10 as an inhibitor of neutrophil elastase, preferably human neutrophil elastase (SEQ ID NO:9).

21 . Use of the polypeptide of any one of claims 1 -6 and conjugate of any one of claims 7-10 as a mitochondria-selective targeting agent.

22. Use of the polypeptide of any one of claims 1 -6 and conjugate of any one of claims 7-10 as a medicament.