Membrane translocation domains and uses thereof

Abstract

Described herein are peptides comprising a membrane translocation domain having one or more cell penetrating peptide motifs, wherein at least one cell penetrating peptide motif has a length of from 3 to 10 amino acid residues and has at least three arginine and / or lysine residues; or wherein at least one cell penetrating peptide motif has a length of from 3 to 10 amino acid residues and has at least two arginine and / or lysine residues, and at least one other cell penetrating peptide motif has a length of from 2 to 8 amino acid residues and has at least two hydrophobic residues. Compositions comprising cargo motifs bound to the disclosed peptides are also disclosed. Also disclosed are methods of delivering a cargo moiety into a cell comprising contacting the cell with a peptide as disclosed herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 320,978, filed on March 17, 2022, which is incorporated herein by reference in its entirety.

[0003] Government Support Statement

[0004] This invention was made with government support under Grant No. GM122459 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Reference to sequence listings submitted electronically

[0006] The application contains a sequence listing, which is submitted electronically via EFS-Web in the form of a sequence listing in ASCII format, with a file name of "103361-223WO1_ST26.xml", a creation date of March 17, 2023, and a size of 392417 bytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background Art

[0007] The efficient delivery of biomolecules (e.g., peptides, proteins, and nucleic acids) to the interior of cells (e.g., the cytoplasm and nucleus) will open the door to a new generation of therapeutics capable of treating many currently intractable diseases. Over the past few decades, researchers have explored a variety of delivery modes, including cell-penetrating peptides for various biomolecular cargoes (Muhammad MN, et al. (2021) Cell penetrating peptides: A versatile vector for co-delivery of drug and genes in cancer. J. Contr. Rel. 330: 1220-1228; Heitz, F., et al. (2009) Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 157: 195-206), bacterial toxins (Pavlik, BJ, et al. (2017) Repurposed bacterial toxins for human therapeutics. Curr. Topics Peptide Protein Res. 18: 1-15; Shorter SA, et al. (2017) The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery. Expert Op. Drug Deliv.14(5):685-696), viruses (Wang, D., et al. (2019) Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov.18:358-378), polyplexes (Ita, K. (2020) Polyplexes for gene and nucleic acid delivery: Progress and bottlenecks, Eur. J. Pharm. Sci.150:105358), liposomes (Pattni, BS, et al. (2015) New Developments in Liposomal Drug Delivery. Chem. Rev.115(19):10938-66) and nanoparticles (Mitchell, MJ, et al. (2021) Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20: 101-124). Although some delivery methods have demonstrated clinical success (e.g., liposome delivery of mRNA vaccines (Kim, EM, et al. (2021) Liposomes: Biomedical Applications. Chonnam Med. J. 57 (1): 27-35)), viral delivery of gene therapy (Wang, D., supra) and anticancer agents based on bacterial toxins (Pavlik BJ, supra), there are still significant limitations. For example, virus-, liposome-, and nanoparticle-based delivery systems are often sequestered by the liver and spleen, which limits their biodistribution to other disease tissues, while non-viral delivery systems also face the major challenge of endosomal entrapment (Patra, JK, et al. (2018) Nanobased drug delivery systems: recent developments and future prospects. J. Nanobiotechnol. 16, 71; Pei D., et al. (2019) Overcoming Endosomal Entrapment in Drug Delivery. Bioconjug. Chem. 30(2): 273-283).

[0008] A family of cyclic CPPs has been discovered that enter mammalian cells through endocytosis and efficiently escape early endosomes into the cytoplasm through a vesicle budding-and-collapse mechanism (Qian Z., et al. (2013) Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs. ACS Chem. Biol. 8(2): 423-31; Qian, Z., et al. (2016) Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides. Biochem. 55(18): 2601-2612; Sahni, A., et al. (2020) Cell-Penetrating Peptides Escape the Endosome by Inducing Vesicle Budding and Collapse. ACS Chem. Biol. 15(9): 2485-2492). These cyclic CPPs have been shown to be very effective for the cytoplasmic delivery of the main drug modes (e.g., small molecules, peptides, proteins, and nucleic acids) in vitro and in vivo. However, cyclic CPPs contain non-proteinogenic amino acids and must be synthesized by chemistry and conjugated with the cargo molecule of interest, which makes them less suitable for protein delivery, because the site-specific conjugation of proteins with other entities is still a major challenge. In order to overcome this limitation, short CPP motifs (e.g., Arg-Arg-Arg-Arg-Trp-Trp-Trp or R4W3) (SEQ.ID.NO.: 133) are genetically inserted into the surface loop of the target protein, making the latter cell permeable (Chen, K., et al. (2020) Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops.ACS Chem.Biol.15(9): 2568-2576). Although this method avoids the need for post-translational modification of proteins, it still has many disadvantages. First, this approach usually requires the availability of structural information of the target protein in order to identify the correct surface loops for CPP insertion. Second, even if structural information is already available, one may still need to experimentally test several different insertion sites as well as multiple CPP sequences to find the optimal combination of insertion site and CPP sequence.For some proteins, it may not be possible to generate insertion mutants without compromising their stability and / or activity. Finally, each target protein is unique in structure and properties and requires different engineering / optimization activities. What is needed are efficient, versatile protein delivery systems that can eliminate some or all of the above disadvantages. The compositions and methods disclosed herein address these needs and others. Summary of the invention

[0009] Compounds, compositions, methods for preparing and using such compounds and compositions are disclosed herein. On the one hand, peptides comprising a membrane translocation domain having one or more cell-penetrating peptide motifs are disclosed, wherein at least one cell-penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least three arginine and / or lysine residues; or wherein at least one cell-penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least two arginine and / or lysine residues, and at least one other cell-penetrating peptide motif has a length of 2 to 8 amino acid residues and has at least two hydrophobic residues. Compositions comprising cargo motifs bound to the disclosed peptides are also disclosed. Methods for delivering cargo moieties into cells are also disclosed, comprising contacting cells with peptides as disclosed herein.

[0010] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention are apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the invention.

[0012] Figure 1 . Structures of WT FN3 and MTD1-10. The FN3 structure was generated from PDB file 1ttg, and the structure of MTD1-10 was predicted by Phyre2. The inserted CPP motif of MTD1-10 is highlighted in black.

[0013] Figure 2A and Figure 2B .Expression and purification of MTD4. Figure 2A FPLC chromatogram showing the elution of MTD4 from a Ni-NTA column (MTD4 is eluted as a broad peak); Figure 2B Figure 2 is an SDS-PAGE showing the expression level and different fractions during purification on a Ni-NTA column. L, molecular weight marker; U, crude lysate of uninduced cells; I, crude lysate of IPTG-induced cells; CL, crude cell lysate after centrifugation; FT, flow-through fraction; 1-10, Ni-NTA column elution fraction (strong band is MTD4).

[0014] Figure 3A to Figure 3I. with 5 μM FN3 TMR (Figure 3A), MTD2 TMR (Figure 3B), and MTD4 TMR (Figure 3C), MTD4 TMR (Figure 3D), MTD6 TMR (Figure 3E) and MTD7 TMR (Figure 3F), MTD8 TMR (Figure 3G), MTD9 TMR (Figure 3H) or MTD10 TMR (FIG. 3I) Live cell confocal microscopy images of HeLa cells after 2 hours of incubation. All images were acquired under the same conditions (including laser power and signal amplification).

[0015] Figure 4 . Overall cellular uptake efficiency of TMR-labeled protein domains as measured by flow cytometry. The values ​​represent the mean fluorescence intensity of treated cells.

[0016] Figure 5 Western blot analysis of total pY levels in NIH3T3 cells after 4 h of treatment with increasing concentrations of MTD4-PTP1B (0-5 μM). Anti-pY antibody 4G10 was used for pY detection. The same membrane was reblotted with anti-GAPDH antibody to ensure equal loading. L, molecular weight marker.

[0017] Figure 6A to Figure 6B. MTD4-RBDV (Figure 6A), MTD4-NS1 (Figure 6B) and NS1-MTD4 ( Figure 6C ) on the cell viability of various cancer cell lines. Cells were treated with the indicated concentrations of fusion protein or vehicle (PBS) at 37°C for 72 hours. The cells were then incubated with Cell Titer Glo solution for 15 minutes and luminescence was measured. The reported viability values ​​are relative to the viability values ​​of cells treated with vehicle (PBS).

[0018] Figure 7 .Inhibition of Ras-Raf interaction by MTD4-RBDV and MTD4-NS1 in HEK293T cells. Cells were transfected with pEF-RLUC8-L15-KrasG12V (or G12D) and pEF-CRAFRBD(1-149)-L15-GFP and incubated at 37°C for 24 h. Cells were then treated with the indicated concentrations of fusion proteins for 20-24 h. BRET signals were measured after adding 10 μM coelenterazine 400a as a substrate.

[0019] Figures 8A-8B. Western blot analysis. Effects of MTD4-RBDV (Figure 8A) and MTD4-NS1 (Figure 8B) on Akt and MEK phosphorylation in MiaPaCa-2 cells. GAPDH was used as a loading control.

[0020] Fig. 9 MTD4-RBDV and MTD4-NS1 induce apoptosis in lung cancer H358 cells. Approximately 10,000 cells were treated with the indicated concentrations of MTD4-RBDV or MTD4-NS1 in the presence of 10% FBS for 24 h and then stained with Alexa Fluor 488-Annexin V and propidium iodide staining followed by flow cytometric analysis.

[0021] 10A-10D . Live cell confocal microscopy images of HEK293T cells after 6 hours of incubation with buffer ( FIG. 10A ), 10 μM GFP11 ( FIG. 10B ), CPP12-GFP11 ( FIG. 10C ), or MTD4-GFP11 ( FIG. 10D ).

[0022] Fig.11 : Dose-dependent induction of luciferase activity in HEK293T cells transiently expressing LgBit by HiBit or HiBit conjugated to different delivery vehicles. The y-axis values ​​are all relative to untreated cells (without HiBit) and represent the mean ± SD of three independent experiments.

[0023] Figures 12A to 12C: Confocal microscopy images of live cells after incubation of HeLa cells with buffer (Figure 12A), 5μM SEP (Figure 12B) or 5μM MTD4-SEP (Figure 12C) for 2 hours. Top panel, nuclear staining with Hoechst; bottom panel, fluorescence of SEP.

[0024] Figures 13A to 13B: Dephosphorylation of pY protein in HEK293T cells by MTD4-PTP1B. (Figure 13A) Western blot analysis of cell lysates after cells were treated with buffer PTP1B (WT; 5 μM) or increasing concentrations of MTD4-PTP1B (0M-5 μM) for 6 hours. (Figure 13B) Coomassie blue staining of the SDS-PAGE gel in (Figure 13A) showed that similar amounts of protein samples were loaded on all lanes.

[0025] Fig.14: Biodistribution of MENC protein in different organs of transgenic mice. The green and red channels correspond to the signals from EGFP and m-cherry, respectively. The fluorescence was monitored in different tissues of mice euthanized after 3 h (EGFP) and 48 h (mCherry).

[0026] Figures 15A-15B: SDS-PAGE analysis of MTD4 (Figure 15A) and FN3 (Figure 15B) after incubation with human serum for different time periods (0-24 hours). The protein bands in the box correspond to MTD4 / FN3 and its degradation products. DETAILED DESCRIPTION

[0027] The present invention may be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0028] Before disclosing and describing the compounds, compositions, articles, systems, devices and / or methods of the present invention, it should be understood that, unless otherwise stated, they are not limited to specific synthetic methods, or, unless otherwise stated, they are not limited to specific reagents, as they can of course vary. It should also be understood that the terms used herein are only for the purpose of describing specific aspects and are not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0029] All publications mentioned herein are incorporated herein by reference, to disclose and describe methods and / or materials in conjunction with the cited publications. The publications discussed herein are provided only because they are disclosed before the filing date of the present application. Anything herein should not be construed as admitting that the present invention is not entitled to be published in advance due to previous inventions. In addition, the release date provided herein may be different from the actual release date, which may require independent confirmation.

[0030] definition

[0031] Throughout this specification, the terms "about" and / or "approximately" may be used in conjunction with numerical values ​​and / or ranges. The term "about" should be understood to refer to those values ​​that are close to the recited values ​​as well as the recited values.

[0032] Throughout this specification, numerical ranges of certain amounts are provided. It should be understood that these ranges include all values ​​and subranges therein. Therefore, the range "from 50 to 80" includes all possible values ​​therein (e.g., 50, 51, 52, 53, 54, 55, 56, etc.) and all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). In addition, all values ​​in a given range can be the endpoints of the range thus covered (e.g., the range 50-80 includes a range with endpoints, such as 55-80, 50-75, etc.).

[0033] The term "a / an" refers to one or more of the entities; for example, "Polypeptide Conjugate" refers to one or more Polypeptide Conjugates or at least one Polypeptide Conjugate. Thus, the terms "a / an", "one or more" and "at least one" are used interchangeably herein. In addition, reference to a "Polypeptide Conjugate" by the indefinite article "a / an" does not exclude the possibility that more than one Polypeptide Conjugate is present, unless the context clearly requires that only one Polypeptide Conjugate is present.

[0034] As used herein, the term "adjacent" refers to two adjacent amino acids connected by a covalent bond. "Adjacent" can also be used interchangeably with "contiguous".

[0035] The term "carrier" refers to a compound, composition, substance or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity or any other characteristic of a compound or composition to achieve its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects to a subject.

[0036] As used herein, "treat", "treating", "treatment" and variations thereof refer to any administration of a Polypeptide Conjugate of the present disclosure that partially or completely alleviates, ameliorates, prevents, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of a disease or condition as described herein.

[0037] As used herein, "therapeutically effective" refers to an amount of a Polypeptide Conjugate of the present disclosure or a complex thereof that can deliver an amount of a therapeutic nucleic acid that confers a therapeutic effect on a patient.

[0038] As used herein, "cell penetrating peptide" or "CPP" refers to any peptide that can penetrate a cell membrane. As used herein, "cyclic cell penetrating peptide" or "cCPP" refers to any cyclic peptide that can penetrate a cell membrane.

[0039] As used herein, "linker" or "L" refers to a moiety that covalently attaches two or more components of the polypeptide conjugates disclosed herein (e.g., a linker can covalently attach a CPP and a group that binds to a nucleic acid sequence through electrostatic interactions (i.e., P)). In some embodiments, the linker can be a natural or non-natural amino acid or polypeptide. In other embodiments, the linker is a synthetic compound containing two or more suitable functional groups suitable for binding, for example, a CPP and (independently) P. In some embodiments, the linear length of the linker (excluding branching atoms or substituents) is about 3 to about 100 (e.g., about 3 to about 20) atoms. In some embodiments, the linker provides about 1 to about 20 atoms between the two groups to which it is attached. to about distance.

[0040] As used herein, "polypeptide" refers to a string of at least two amino acids attached to each other by peptide bonds. There is no upper limit to the number of amino acids that can be included in a polypeptide. In addition, a polypeptide may include non-natural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide.

[0041] As used herein, "monomer" refers to an amino acid residue in a polypeptide. In some embodiments, an amino acid monomer is divalent. In other embodiments, if the amino acid monomer is further substituted, the amino acid monomer may be trivalent. For example, a cysteine ​​monomer may independently form a peptide bond at the N-terminus and the C-terminus, and may also form a disulfide bond.

[0042] As used herein, "amino acid analogs" or "analogs" (e.g., "arginine analogs," "lysine analogs," or "histidine analogs") refer to amino acid variants that retain at least one function of an amino acid, such as the ability to bind to an oligonucleotide via electrostatic interactions. Such variants may have elongated or shorter side chains (e.g., through one or more -CH2- groups, which retain the ability to bind to an oligonucleotide via electrostatic interactions, or, the modification may improve the ability to bind to an oligonucleotide via electrostatic interactions). For example, an arginine analog may contain an additional methylene or ethylene group between the backbone and the guanidine / guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, C1-haloalkyl, C2-haloalkyl, amine, guanidine, etc.). Amino acid analogs may be monovalent, divalent, or trivalent.

[0043] Throughout this specification, peptides and amino acid monomers are depicted as electrically neutral species. It should be understood that such species may carry a positive or negative charge, depending on the conditions. For example, at pH 7, the N-terminus of an amino acid is protonated and carries a positive charge (-NH3 +), while the C-terminus of the amino acid is deprotonated and carries a negative charge (-CO2 - ). Similarly, the side chains of some amino acids can have either a positive or negative charge.

[0044] Each amino acid can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homologue of a natural amino acid, because it has a structure similar to that of a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Therefore, as used herein, the term "amino acid" refers to natural and non-natural amino acids and their analogs and derivatives. Examples of suitable amino acids include, but are not limited to, alanine, allo-isoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. Amino acid analogs encompass amino acid analogs that have similar structures but are not identical to amino acids, for example, due to modifications to the side chains or main chains on the amino acids. Such modifications can increase the hydrophobicity of the side chain, including extending the side chain by one or more hydrocarbons, or increasing the solvent accessible surface area (such as SASA described herein) of an amino acid having an aromatic ring on the side chain, for example, by conjugating a second aromatic ring or increasing the size of the aromatic ring. Derivatives of amino acids encompass natural and non-natural amino acids that have been modified (e.g., by substitution) to include a hydrophobic group as described herein. For example, derivatives of lysine include lysine whose side chain is substituted by an alkylcarboxamide group. These and other names are listed in Table 1 along with the abbreviations used herein.

[0045] Table 1. Amino acid abbreviations

[0046]

[0047]

[0048] *Single letter abbreviations: When shown herein in upper case letters, they indicate the L-amino acid form, and when shown herein in lower case letters, they indicate the D-amino acid form.

[0049] "Alkyl" or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain radical having from one to twelve carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups containing any number of carbon atoms from 1 to 12 are included. Alkyl groups containing up to 12 carbon atoms are C1-C 12Alkyl groups, containing up to 10 carbon atoms, are C1-C 10 Alkyl, an alkyl containing up to 6 carbon atoms is a C1-C6 alkyl, and an alkyl containing up to 5 carbon atoms is a C1-C5 alkyl. C1-C5 alkyl includes C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl and C1 alkyl (i.e. methyl). C1-C6 alkyl includes all of the above with respect to C1-C5 alkyl, and also includes C6 alkyl. C1-C 10 Alkyl includes all of the above-mentioned C1-C5 alkyl and C1-C6 alkyl parts, and also includes C7, C8, C9 and C 10 Similarly, C1-C 12 Alkyl includes all of the aforementioned moieties, and also includes C 11 and C 12 Alkyl. C1-C 12 Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless specifically stated otherwise in the specification, the alkyl group may be optionally substituted.

[0050] "Alkylene" or "alkylene chain" refers to a fully saturated straight or branched divalent hydrocarbon chain radical having from one to forty carbon atoms. 40 Non-limiting examples of alkylene include ethylene, propylene, n-butylene, pentylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted as described herein.

[0051] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 12 are included. Alkenyl groups containing up to 12 carbon atoms are C2-C 12 Alkenyl, alkenyl containing up to 10 carbon atoms is C2-C 10 Alkenyl, alkenyl groups containing up to 6 carbon atoms are C2-C6 alkenyl, and alkenyl groups containing up to 5 carbon atoms are C2-C5 alkenyl. C2-C5 alkenyl includes C5 alkenyl, C4 alkenyl, C3 alkenyl, and C2 alkenyl. C2-C6 alkenyl includes all of the above with respect to C2-C5 alkenyl, and also includes C6 alkenyl. C2-C 10 Alkenyl includes all of the above-mentioned moieties regarding C2-C5 alkenyl and C2-C6 alkenyl, and also includes C7, C8, C9 and C 10 Similarly, C2-C 12 Alkenyl includes all of the aforementioned moieties, and also includes C 11and C 12 Alkenyl. C2-C 12 Non-limiting examples of alkenyl groups include ethenyl / vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4- The alkyl groups may be optionally substituted, unless otherwise specifically stated in the specification, or alkyl groups may be optionally substituted.

[0052] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain radical having from two to forty carbon atoms and having one or more carbon-carbon double bonds. 40 Non-limiting examples of alkenylene include ethenylene (-CH=CH-), propenylene, butenylene, etc. Unless stated otherwise specifically in the specification, an alkenylene chain may be optionally substituted.

[0053] "Alkynyl" or "alkynyl group" refers to a straight or branched hydrocarbon chain radical having from two to twelve carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl groups containing any number of carbon atoms from 2 to 12 are included. Alkynyl groups containing up to 12 carbon atoms are C2-C 12 Alkynyl. Alkynyl containing up to 10 carbon atoms is C2-C 10 Alkynyl, an alkynyl group containing up to 6 carbon atoms is a C2-C6 alkynyl, and an alkynyl group containing up to 5 carbon atoms is a C2-C5 alkynyl. C2-C5 alkynyl includes C5 alkynyl, C4 alkynyl, C3 alkynyl, and C2 alkynyl. C2-C6 alkynyl includes all of the above with respect to C2-C5 alkynyl, and also includes C6 alkynyl. C2-C 10Alkynyl includes all of the above-mentioned moieties regarding C2-C5 alkynyl and C2-C6 alkynyl, and also includes C7, C8, C9 and C 10 Similarly, C2-C 12 Alkynyl includes all of the aforementioned moieties, and also includes C 11 and C 12 Alkynyl. C2-C 12 Non-limiting examples of alkenyl include ethynyl, propynyl, butynyl, pentynyl, etc. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted.

[0054] "Alkyne" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain radical having from two to forty carbon atoms and having one or more carbon-carbon triple bonds. 40 Non-limiting examples of alkynylene groups include ethynylene (-C≡C-), propynylene, etc. Unless stated otherwise specifically in the specification, an alkynylene chain may be optionally substituted.

[0055] "Aryl" refers to a hydrocarbon ring system containing hydrogen, 6 to 40 carbon atoms and at least one aromatic ring. For the purposes of this disclosure, an aryl group can be a monovalent or divalent group (excluding substituents), which can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and can include fused or bridged ring systems. Aryl groups include, but are not limited to, those derived from aceanthrene, acenaphthylene, acephenanthrene, anthracene, azulene, benzene, The aryl groups include fluoranthene, fluorene, asymmetric indacene, symmetric indacene, indane, indene, naphthalene, phenanthene, phenanthene, pyrene and triphenylene. In certain embodiments, when used as a joint or as a part of a joint, the aryl group can be divalent. Unless otherwise specifically stated in the specification, the aryl group can be optionally substituted.

[0056] As used herein, "aromatic" refers to an unsaturated cyclic molecule having 4n+2π electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated cyclic molecule that does not fall within the definition of aromatic.

[0057] "Carbocyclyl", "carbocyclic ring" or "carbocycle" refers to a ring structure in which each of the atoms forming the ring is carbon. The carbocycle may contain 3 to 20 carbon atoms in the ring. Carbocycles include aryl and cycloalkyl groups as well as fully unsaturated, partially unsaturated and fully saturated rings. In some embodiments, when used as a joint or as part of a joint, the carbocyclyl group may be divalent. Unless otherwise specifically stated in the specification, the carbocyclyl group may be optionally substituted.

[0058] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon group having 3 to 40 carbon atoms and at least one ring, wherein the ring is composed only of carbon and hydrogen atoms, and the ring may include a fused or bridged ring system. For the purposes of this disclosure, a cycloalkyl group may be a monovalent or divalent group (excluding substituents). Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo [2.2.1] heptyl, etc. In some embodiments, when used as a joint or as part of a joint, a cycloalkyl group may be divalent. Unless otherwise specifically stated in the specification, a cycloalkyl group may be optionally substituted.

[0059] "Cycloalkenyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 40 carbon atoms, at least one ring and one or more carbon-carbon double bonds, wherein the ring is composed only of carbon and hydrogen atoms, and the ring may include a fused or bridged ring system. For purposes of the present invention, cycloalkenyl may be a monovalent or divalent group (excluding substituents). Monocyclic cycloalkenyl groups include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc. Polycyclic cycloalkenyl groups include, for example, bicyclo [2.2.1] hept-2-enyl, etc. In certain embodiments, when used as a joint or as a part of a joint, the cycloalkenyl group may be divalent. Unless otherwise specifically stated in the specification, the cycloalkenyl group may be optionally substituted.

[0060] "Cycloalkynyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 40 carbon atoms, at least one ring and one or more carbon-carbon triple bonds, wherein the ring is composed only of carbon and hydrogen atoms, and the ring may include a fused or bridged ring system. For purposes of the present invention, cycloalkynyl may be a monovalent or divalent group (excluding substituents). Monocyclic cycloalkynyl includes, for example, cycloheptynyl, cyclooctynyl, etc. In certain embodiments, when used as a joint or as a part of a joint, the cycloalkynyl group may be divalent. Unless otherwise specifically stated in the specification, the cycloalkynyl group may be optionally substituted.

[0061] "Heterocyclyl", "heterocyclic ring" or "heterocycle" refers to a stable 3- to 20-membered aromatic ring radical consisting of two to twelve carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For the purposes of the present invention, a heterocyclyl group may be a monovalent or divalent radical (excluding substituents). Heterocyclyl or heterocycle includes heteroaryl as defined below. Unless otherwise specifically stated in the specification, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged ring system; and the nitrogen, carbon or sulfur atoms in the heterocyclyl group may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl group may be partially or fully saturated. Examples of such heterocyclic groups include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl. In some embodiments, when used as a joint or as part of a joint, the heterocyclic group can be divalent. Unless otherwise specifically stated in the specification, the heterocyclic group can be optionally substituted.

[0062] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing hydrogen atoms, one to fourteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, a heteroaryl group can be a monovalent or divalent group (excluding substituents), and can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azacycloheptatrienyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothienyl / benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophene yl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinetrienyl, oxazolyl, oxiranyl, 1-oxopyridinyl, 1-oxopyrimidinyl, 1-oxopyrazinyl, 1-oxopyridazinyl , 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolyl, quinuclidinyl, isoquinolyl, tetrahydroquinolyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thiophenyl (i.e., thienyl). In certain embodiments, when used as a joint or as part of a joint, a heteroaryl group can be divalent. Unless otherwise specifically stated in the specification, a heteroaryl group can be optionally substituted.

[0063] The term "ether" as used herein refers to a straight or branched chain divalent radical moiety -[(CH2) m -O-(CH2) n ] z -, wherein each of m, n and z is independently selected from 1 to 40. Examples include, but are not limited to, polyethylene glycol. Unless otherwise specifically stated in the specification, ethers may be optionally substituted.

[0064] As used herein, the term "substituted" means that at least one hydrogen atom in any of the above groups (i.e., alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl and / or ether) is replaced by a bond to a non-hydrogen atom, such as, but not limited to, halogen atoms such as F, Cl, Br and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means that one or more hydrogen atoms in any of the above groups are replaced by a higher order bond (e.g., double or triple bond) to a heteroatom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imide, oxime, hydrazone, and nitrile. For example, "substituted" includes one or more hydrogen atoms in any of the above groups replaced by -NR g R h 、-NR g C(=O)R h 、-NR g C(=O)NR g R h 、-NR g C(=O)OR h 、-NR g S02R h 、-OC(=O)NR g R h 、-OR g 、-SR g 、-SOR g 、-SO2R g 、-OSO2R g 、-SO2OR g , =NSO2R g and -SO2NR g R h "Substituted" also means that one or more hydrogen atoms in any of the above groups are replaced by -C(=O)R g 、-C(=O)OR g 、-C(=O)NR g R h 、-CH2SO2R g 、-CH2SO2NR g R h Replace. In the above, R g and Rh The same or different, and independently be hydrogen, alkyl, alkenyl, alkynyl, alkoxyl, alkylamino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclic radical, N-heterocyclic radical, heterocyclic radical alkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substituted" further means that one or more hydrogen atoms in any of the above-mentioned groups are replaced by the key with amino, cyano, hydroxyl, imino, nitro, oxo, thio, halo, alkyl, alkenyl, alkynyl, alkoxyl, alkylamino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclic radical, N-heterocyclic radical, heterocyclic radical alkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl groups. In addition, each of the above-mentioned substituents can also be optionally substituted by one or more of the above-mentioned substituents. Furthermore, one skilled in the art will recognize that "substituted" also encompasses situations in which one or more atoms on any of the above groups are substituted with a substituent listed in this paragraph, and the substituent forms a covalent bond with the CPP, P, or L. For example, in certain embodiments, any of the above groups can be substituted at the first position with a carboxylic acid (i.e., -C(=O)OH) that forms an amide bond with lysine in the CPP, or the group can be substituted at the second position with a thiol group that forms a disulfide bond with cysteine ​​(or an amino acid analog having a thiol group).

[0065] As used herein, the term "subject" refers to the target of administration, such as a subject. Therefore, the subject of the method disclosed herein can be a vertebrate, such as a mammal, fish, bird, reptile or amphibian. Alternatively, the subject of the method disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cattle, cat, guinea pig, fish, bird, rodent or fruit fly. The term does not indicate a specific age or gender. Therefore, adult and neonatal subjects and fetuses, whether male or female, will be included. In some instances, the subject is a mammal. A patient refers to a subject suffering from a disease or condition. The term "patient" includes human and veterinary subjects. In some instances of the disclosed method, the subject has been diagnosed with the need for treatment of cancer, autoimmune disease and / or inflammation before the application step. In some instances of the disclosed method, the subject has been diagnosed with cancer before the application step. The term subject also includes cells, such as animal cells, for example human cells.

[0066] As used herein, the term "treatment" refers to the medical management of patients intended to cure, improve or stabilize a disease, pathological condition or condition. The term includes active treatment, i.e., treatment specifically for improving a disease, pathological condition or condition, and also includes etiological treatment, i.e., treatment for the removal of the cause of the associated disease, pathological condition or condition. In addition, the term also includes palliative treatment, i.e., treatment intended to relieve symptoms rather than cure a disease, pathological condition or condition; and supportive treatment, i.e., treatment for supplementing another specific therapy for improving an associated disease, pathological condition or condition. In some instances, the term encompasses any treatment of a subject (including, mammals (e.g., humans)), and includes: (i) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (ii) inhibiting the disease, i.e., curbing its development; or (iii) alleviating the disease, i.e., causing the disease to regress.

[0067] As used herein, the term "prevent" or "preventing" means to exclude, circumvent, avoid, foreclose, stop or hinder something from happening, especially by anticipatory action.

[0068] As used herein, the term "diagnosed" means that a physical examination has been performed by a skilled person (e.g., a physician) and found to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. For example, "diagnosed with cancer" means that a physical examination has been performed by a skilled person (e.g., a physician) and found to have a condition that can be diagnosed or treated by a compound or composition that can treat or prevent cancer. As a further example, "diagnosed as needing treatment or prevention of cancer" means that a physical examination has been performed by a skilled person (e.g., a physician) and found to have a condition characterized by cancer or other diseases, where treating or preventing cancer would be beneficial to the subject.

[0069] As used herein, the phrase "identified as requiring treatment of a condition" and the like refers to selecting a subject based on the need to treat the condition. For example, based on an early diagnosis by a technician, a subject may be identified as requiring treatment of a condition (e.g., a condition associated with cancer) and then receive treatment for the condition. In some instances, it is contemplated that the identification may be performed by a person different from the person making the diagnosis. In some instances, it is also contemplated that administration may be performed by a person who subsequently performs the administration.

[0070] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections, such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration may be continuous or intermittent. In some instances, the formulation may be administered therapeutically; that is, administered to treat an existing disease or condition. In some instances, the formulation may be administered prophylactically; that is, administered to prevent a disease or condition.

[0071] As used herein, the term "contacting" refers to bringing a disclosed compound and a target (e.g., a cell, a target receptor, a transcription factor, or other biological entity) together so that the compound can affect the activity of the target directly (i.e., by interacting with the target itself) or indirectly (i.e., by interacting with another molecule, cofactor, factor, or protein on which the target activity depends).

[0072] As used herein, the terms "effective amount" and "amount effective" refer to an amount sufficient to achieve a desired result or to produce an effect on an undesirable condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to produce an effect on an undesirable symptom but generally insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the specific composition employed; the patient's age, weight, general health, sex and diet; the time of administration; the route of administration; the excretion rate of the specific compound employed; the duration of treatment; the drugs used in combination or concurrently with the specific compound employed and similar factors well known in the medical field. For example, it is within the skill of the art to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. If necessary, the effective daily dose can be divided into multiple doses for administration purposes. Therefore, a single dose composition may contain such an amount or a submultiple thereof to constitute the daily dose. In the case of any contraindications, the dose may be adjusted by the individual physician. Dosages may vary, and may be administered in one or more doses per day, for one or more days. Guidance on appropriate dosages for a given class of drug may be found in the literature. In some instances, a formulation may be administered in a "prophylactically effective amount"; that is, an amount effective to prevent a disease or condition.

[0073] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, ie, does not cause unacceptable levels of undesired biological effects or interact in a deleterious manner.

[0074] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders reconstituted as sterile injectable solutions or dispersions before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc., it is ensured that microorganisms are prevented. It may also be necessary to include isotonic agents, such as sugars, sodium chloride, etc. The absorption of injectable drug forms can be extended by including agents that delay absorption (such as aluminum monostearate and gelatin). Injectable reservoir forms are prepared by forming a microencapsulated matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). The drug release rate can be controlled according to the ratio of the drug to the polymer and the properties of the specific polymer used. Reservoir injectable formulations can also be prepared by embedding the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by filtering through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use. Suitable inert carriers can include sugars such as lactose.

[0075] As used in the specification and the appended claims, a residue of a chemical substance refers to the portion of the chemical substance resulting from a particular reaction scheme or subsequent formulation or chemical product, whether or not that portion is actually obtained from the chemical substance. Thus, an amino acid residue in a peptide or protein refers to one or more -OC(O)CH(R)NH- units in the peptide or protein.

[0076] As used herein, the symbol (hereinafter may be referred to as a "point of attachment bond") represents a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other is not depicted as being attached to the point of attachment bond. For example, Indicates that the chemical entity "XY" is bonded to another chemical entity through an attachment point bond. In addition, the specific attachment point to the chemical entity not depicted can be specified by inference. For example, the compound CH3-R 3 , where R 3 H or It shows that when R 3 When "XY", the attachment point key is the same as R 3 Depicted as the same bond to which CH3 is bonded.

[0077] Unless otherwise specified, formulas with chemical bonds shown only as solid lines, rather than wedges or dashed lines, contemplate every possible isomer, such as each enantiomer and diastereomer, and mixtures of isomers, such as racemic or scalemic mixtures. The compounds described herein may contain one or more asymmetric centers, and thus may produce diastereomers and optical isomers. Unless otherwise specified, the compounds and compositions disclosed herein include all such possible diastereomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers as well as isolated specific stereoisomers are also included. In synthetic procedures for preparing such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the product of such procedures may be a mixture of stereoisomers.

[0078] Many organic compounds exist in optically active forms, with the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to its chiral center. The prefixes d and l or (+) and (-) are used to indicate the rotation sign of the compound to plane polarized light, wherein (-) or means that the compound is left-handed. Compounds prefixed with (+) or d are right-handed. For a given chemical structure, these compounds (called stereoisomers) are identical, except that they are non-superimposable mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of these isomers are generally referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many compounds described herein may have one or more chiral centers, and therefore may exist in different enantiomeric forms. If desired, chiral carbon may be represented by an asterisk (*). When the bonds to a chiral carbon are depicted as straight lines in a disclosed formula, it is understood that both the (R) and (S) configurations of the chiral carbon and therefore both enantiomers and mixtures thereof are encompassed by the formula. As used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon may be depicted as a wedge (bond to an atom above the plane) and the other may be depicted as a series or wedge of short parallel lines (bonded to an atom below the plane). The Cahn-Inglod-Prelog system may be used to assign the (R) or (S) configuration to a chiral carbon.

[0079] The compounds described herein contain atoms in both natural isotopic abundance and unnatural abundance. The disclosed compounds may be isotopically labeled or isotopically substituted compounds, which are identical to those described, but where one or more atoms are replaced by an atom having an atomic mass or mass number that is comparable to the atomic mass or mass number commonly found in nature. Examples of isotopes that may be incorporated into the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as respectively 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 Cl. The compound further includes its prodrug, and pharmaceutically acceptable salts of the compound or the prodrug containing the aforementioned isotopes and / or other isotopes of other atoms are also within the scope of the present invention. Certain isotopically labeled compounds, for example, incorporating radioactive isotopes such as 3 H and 14 C, can be used in drug and / or substrate tissue distribution assays. Tritiated isotopes, i.e. 3H and carbon-14 isotopes, i.e. 14 C is particularly preferred due to its ease of preparation and detectability. In addition, heavier isotopes such as deuterium (i.e. 2 H) substitution may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. Isotopically labeled compounds and prodrugs thereof can generally be prepared by carrying out the following procedure by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0080] Components for preparing the compositions disclosed herein and the compositions themselves used in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and it should be understood that when the combinations, subsets, interactions, groups, etc. of these materials are disclosed, although specific references to the individual and collective combinations and arrangements of these compounds cannot be explicitly disclosed, each is specifically considered and described herein. For example, if a specific compound is disclosed and discussed, and many modifications that can be made to many molecules containing these compounds are discussed, each combination and arrangement of the compound and possible modifications is specifically considered, unless the contrary is specifically indicated. Therefore, if the categories of molecules A, B, and C and the categories of molecules D, E, and F are disclosed, as well as examples of combined molecules AD, each molecule is a combination of meanings considered individually and collectively, even if each molecule is not listed separately, and AE, AF, BD, BE, BF, CD, CE, and CF are also considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Therefore, for example, the subgroups of AE, BF, and CE will be considered to be disclosed. This concept applies to all aspects of the present application, including but not limited to steps in the method for preparing and using the compositions disclosed herein. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein.

[0081] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0082] Composition

[0083] Disclosed are cell permeable peptides and compositions comprising them, which can provide a universal vehicle for the cytoplasmic delivery of potentially any peptide or protein cargo and other biomolecules. The disclosed composition can have a higher cytoplasmic delivery efficiency and in vivo stability than simple cell permeable peptides. In addition, the disclosed composition can escape the endosome of the cell more completely, and therefore more effectively transport the conjugated cargo out of the endosome than what occurs when there is no disclosed composition. The disclosed composition can also have a wider range of cargo compatibility (basically any peptide or protein). And synthesis can be simpler-the membrane translocation domain gene is fused to the N-terminal, C-terminal or internal position of the cargo protein. In addition, the disclosed composition can have a lower immunogenicity possibility, and the cargo can be delivered to almost any eukaryotic (e.g., mammals and plants) cell, which is different from bacterial toxins, which are limited to cells expressing specific receptors for toxins. The disclosed composition can also have a higher delivery capacity than bacterial toxins, because the delivery capacity of bacterial toxins may be limited by the abundance of receptors on the target cell surface. In a further example, the disclosed composition does not need or contain a cofactor, such as zinc.

[0084] In a specific aspect, disclosed herein are peptides comprising: a membrane translocation domain having one or more cell penetrating peptide motifs, wherein at least one of the cell penetrating peptide motifs is 3 to 10 amino acid residues in length and has at least three arginine and / or lysine residues. Unlike the method of inserting a CPP motif into each target protein, the disclosed compositions and methods relate to engineered membrane translocation domains that can be fused to any target cargo of interest by genetic or synthetic means. Another strategy disclosed herein involves splitting the CPP motif into two halves and inserting them into two different regions of the membrane translocation domain, resulting in greatly improved cytoplasmic delivery efficiency.

[0085] membrane translocation domain

[0086] The membrane translocation domain portion of the disclosed peptide can be any membrane translocation domain, i.e., a peptide sequence that can pass through a lipid bilayer, which has been modified to contain at least one cell penetration motif as described herein. In a preferred embodiment, there are two or three cell penetration motifs in the membrane translocation domain. For example, at least one cell penetration peptide motif may be 3 to 10 amino acid residues in length and have at least three arginine and / or lysine residues, such as 4, 5 or 6 arginine and / or lysine residues. Alternatively, at least one cell penetration peptide motif may be 3 to 10 amino acid residues in length and have at least two arginine and / or lysine residues, and at least one other cell penetration peptide motif may be 2 to 8 amino acid residues in length and have at least two hydrophobic residues. When there are two or more cell penetration peptide motifs, two or more arginine residues and / or lysine residues may be present within a range of 3 to 10 amino acids, and when there is another cell penetration peptide motif, two or more hydrophobic residues may be present within a range of 2 to 8 amino acids. The cell penetration peptide motif may be located at any position in the membrane translocation domain.

[0087] In some examples, the membrane translocation domain can be a human membrane translocation domain, such as type III fibronectin. In a specific example, the membrane translocation domain has at least 90%, at least 95% or at least 97% sequence similarity with SEQ.ID.NO.: 118. In other examples, the membrane translocation domain is a human type III fibronectin having BC, DE, CD and FG loops, and the cell penetrating peptide motif is located in one or more of the BC, DE, CD or FG loops, for example, the cell penetrating peptide motif is located in two of the BC, DE, CD or FG loops, particularly the BC and FG loops. These loops can be defined as having the following sequences BC = AVTVR (SEQ ID NO.: 31); CD = GGNSPVQ (SEQ ID NO.: 32); DE = PGSK (SEQ ID NO.: 33); FG = GRGDSPAS (SEQ ID NO.: 34).

[0088] In other examples, the membrane translocation domain can be any stably folded protein that can be efficiently expressed, preferably in bacteria. Some additional examples of membrane translocation domains are nanobody scaffolds, DARPin scaffolds, and CTPR proteins (shared triangular tetratricopeptide repeats; Acc. Chem. Res. 2021, 54, 4166-4177).

[0089] Cell penetrating peptide motif

[0090] The cell penetrating peptide (CPP) motif may comprise at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, or at least 6 amino acids, more specifically 3 to 8, 3 to 6, 4 to 8, 4 to 6, or 6 to 8 amino acids. In most instances, the CPP motif is substituted into the membrane translocation domain such that the resulting peptide has the same number of amino acids as in the native membrane translocation domain.

[0091] In some examples, at least two, three, four, five, six or seven amino acids of the CPP motif are adjacent arginine residues. In a preferred embodiment, there are three, four or five adjacent arginine residues in the CPP motif. In other examples, the arginine residues are not adjacent in the CPP motif. Each amino acid in the CPP motif can be a natural or non-natural amino acid independently. When such adjacent arginine or lysine residues are CPP motifs, no additional CPP motifs are required, such as those with hydrophobic residues, but such hydrophobic CPP motifs can still be used. When the CPP motif contains two arginine residues, it is preferred that another CPP motif with at least two hydrophobic residues is present within 2 to 8 amino acids.

[0092] In other examples, at least one, at least two, at least three or more amino acids of the CPP motif are hydrophobic amino acids, i.e., have hydrophobic side chains. In some examples, the amino acids with hydrophobic side chains are independently selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylic acid, cyclohexylalanine, norleucine, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, tert-leucine or nicotinoyllysine, each of which is optionally substituted with one or more substituents. In a specific example, each amino acid with a hydrophobic side chain is independently an amino acid with an aromatic side chain. In certain embodiments, the amino acid with an aromatic side chain is 3-benzothienyl-L-alanine, naphthylalanine, phenylglycine, homophenylalanine, phenylalanine, tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. Therefore, in some examples, the amino acid with a hydrophobic side chain is phenylalanine, naphthylalanine, tryptophan or its analog or derivative, naphthylalanine or tryptophan or its analog or derivative. In other examples, the CPP motif further comprises at least one phenylalanine, phenylglycine or histidine, or its analog or derivative.

[0093]

[0094] In some examples, the CPP motif may comprise any combination of at least three adjacent arginines and at least two amino acids having hydrophobic side chains selected from aryl or heteroaryl, wherein the aryl and heteroaryl groups are optionally substituted, and the total number of amino acids in the CPP motif is between 5 and 8 amino acids.

[0095] In some examples, the membrane translocation domain is a human type III fibronectin having BC, DE, CD and FG loops, and the CPP is located in one or more of the BC, DE, CD or FG loops. For example, the CPP motif is located in two of the BC, DE, CD or FG loops. In a specific example, the CPP motif is located in the BC loop and any one of the DE, CD and FG loops, preferably in the BC and FG loops.

[0096] When there are two or more CPP motifs, one CPP motif may be a 3 to 10 amino acid fragment having at least two arginine and / or lysine residues, and the other may be a 2 to 8 amino acid fragment having at least two hydrophobic residues. For example, a membrane translocation domain may have two or more CPPs, and at least one of the motifs is 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues.

[0097] In this example, the membrane translocation domain may be human type III fibronectin having BC, DE, CD and FG loops, and the CPP motif may be located in the BC loop and have 2 to 8 amino acid residues and have at least two hydrophobic amino acid residues, and the CPP motif may be located in the FG loop and have 3 to 10 amino acid residues and have at least three adjacent arginine and / or lysine residues. Alternatively, the CPP motif may be located in the FG loop and have 2 to 8 amino acid residues and have at least two hydrophobic amino acid residues, and the CPP motif may be located in the BC loop and have 3 to 10 amino acid residues and have at least three adjacent arginine and / or lysine residues.

[0098] When the CPP motif contains 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, it may be WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW or WYH. Preferably, the CPP motif is located in the BC loop. Further preferably, the CPP motif is WW, FW, WF, WYW, WWW, WWH, YWW, WYH or YWH.

[0099] A CPP motif having 3 to 10 amino acid residues and having at least three adjacent arginine and / or lysine residues may contain RRR, RRRR (SEQ ID NO: 159), RRRRR (SEQ ID NO: 160). It may also be any combination of arginine and lysine residues. When the CPP motif is located in the FG loop, the length of I may be 3-10 residues and may have any combination of Arg and Lys (and sometimes other non-acidic residues). The CPP motif (e.g., WWWRRRR) (SEQ ID NO: 161) can be selectively cleaved so that some of the Arg / Lys residues move from the FG loop to the BC loop (e.g., WWWR ... RRR (SEQ ID NO: 161), WWWRR ... RR (SEQ ID NO: 161), WWWRRRR ... (SEQ ID NO: 161), etc.); the CPP motif (e.g., WWWRRRR) (SEQ ID NO: 161) can be selectively cleaved so that some of the hydrophobic residues move from the BC loop to the FG loop (e.g., WW ... WRRR, W ... WWRRRR, ...) WWWRRRR (SEQ ID NO: 161). The CPP motif (e.g., WWWRRRR) can be selectively cleaved so that the BC or FG loop contains a combination of hydrophobic residues and positively charged residues (e.g., WWR ... WRRR, WWRR ... WRR, WWRR ... RRW, RRW ... WWRR, etc.) (SEQ ID NO: 161).

[0100] In specific examples, the CPP motif comprises SEQ.ID.NO.:104, 105, 11, 112, 113, 114, 115, 116 or 117.

[0101] In some examples, the CPP motif can be or include any sequence listed in Table 2. In some examples, the cell penetrating peptide can be or include the reverse sequence of any sequence listed in Table 2.

[0102] Table 2. CPP motif sequences

[0103]

[0104]

[0105]

[0106]

[0107] Φ = L-naphthylalanine; φ = D-naphthylalanine; Ω = L-norleucine; r = D-arginine; F = L-phenylalanine; f = D-phenylalanine; q = D-glutamine; X = L-4-fluorophenylalanine; Dap = L-2,3-diaminopropionic acid; Sar, sarcosine; F2Pmp, L-difluorophosphonomethylphenylalanine; Dod, dodecanoyl; Pra, L-propargylglycine; AzK, L-6-azido-2-amino-hexanoic acid; Agp, L-2-amino-3-guanidinopropionic acid; b Cyclization between Pim and Nlys; c Cyclization between Lys and Glu; d Macrocyclization via multicomponent reaction of aziridine aldehydes and isocyanides; e Cyclization between the main chains of Gln residues; f The N-terminal amine and the side chains of two Dap residues bicyclized with Tm; g Three Cys side chains bicyclized with tris(bromomethyl)benzene; h Cyclization via click reaction between Pra and Azk.

[0108] The chirality of the amino acids may be selected to improve the efficiency of cytoplasmic uptake. In some embodiments, at least two of the amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality may be adjacent to each other. In some embodiments, at least three amino acids have alternating stereochemistry relative to each other. In some embodiments, at least three amino acids with alternating chirality relative to each other may be adjacent to each other. In some embodiments, at least two of the amino acids have the same chirality. In some embodiments, at least two amino acids with the same chirality may be adjacent to each other. In some embodiments, at least two amino acids have the same chirality and at least two amino acids have opposite chirality. In some embodiments, at least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Therefore, in some embodiments, adjacent amino acids in cCPP may have any of the following sequences: DL; LD; DLLD (SEQ ID NO: 153); LDDL (SEQ ID NO: 154); LDLLD (SEQ ID NO: 155); DLDDL (SEQ ID NO: 156); DLLDL (SEQ ID NO: 157); or LDDLD (SEQ ID NO: 158).

[0109] Cargo section

[0110] Also described are peptides as disclosed herein, but further comprising a cargo moiety linked to a membrane translocation domain. The cargo moiety can be linked to an amino group (e.g., N-terminus), a carboxylate group (e.g., C-terminus), or the side chains of one or more amino acids in the membrane translocation domain.

[0111] When the cargo moiety is attached to the side chain of an amino acid in the membrane translocation domain, the membrane translocation domain includes an amino acid having a side chain with a suitable functional group to form a covalent bond with the cargo (conjugation), or a side chain that can be modified to provide a suitable functional group to form a covalent bond with the cargo (e.g., conjugation through a linker). In some embodiments, the amino acid on the membrane translocation domain with a side chain suitable for cargo conjugation is a cysteine ​​residue, a glutamic acid residue, an aspartic acid residue, a lysine residue, or a 2,3-diaminopropionic acid residue. In such embodiments, the cargo can be directly conjugated to the side chain of the amino acid (e.g., by forming a disulfide bond with a cysteine ​​residue or an amide bond with a glutamic acid residue or a 2,3-diaminopropionic acid residue) or the cargo can be conjugated to the amino acid side chain through a linker (e.g., PEG).

[0112] The cargo portion may comprise any cargo of interest, such as a linker portion, a detectable portion, a therapeutic portion, a targeting portion, etc., or any combination thereof. In some examples, the cargo portion may comprise one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., a bifunctional linker LC-SMCC); coenzyme A; phospho-coumaramidopropionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic acid (Dap or J); L-β-naphthylalanine; L-pipecolic acid (Pip); sarcosine; trimesic acid; 7-amino-4-methylcoumarin (Amc); fluorescein isothiocyanate (FITC); L-2-naphthylalanine; norleucine; 2-aminobutyric acid; rhodamine B (Rho); dexamethasone (DEX); or a combination thereof.

[0113] Detectable part

[0114] The detectable moiety may comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, UV-Vis labels, near infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelating centers, heavy atoms, radioisotopes, isotope detectable spin resonance labels, paramagnetic moieties, chromophores, or any combination thereof. In some embodiments, the label can be detected without the addition of other reagents.

[0115] In some embodiments, the detectable moiety is a biocompatible detectable moiety, making the compound suitable for use in a variety of biological applications. As used herein, "biocompatible" and "biologically compatible" generally refer to compounds that are generally non-toxic to cells and tissues, along with any metabolites or degradation products thereof, and do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in their presence.

[0116] The detectable moiety may contain a luminophore, such as a fluorescent label or a near infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins; benzoporphyrins; azabenzoporphyrins; naphthoporphyrins; phthalocyanines; polycyclic aromatic hydrocarbons, such as perylene, perylenediamine, pyrene; azo dyes; xanthene dyes; boron dipyrromethene, aza-boron dipyrromethene, cyanine dyes, metal-ligand complexes such as bipyridine, bipyridyl, phenanthroline, coumarin and acetylacetonate of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; azaannulene, squaryl cyanine; 8-hydroxyquinoline, polymethine, luminescence-producing nanoparticles, such as quantum dots, nanocrystals; quinolones; terbium complexes; inorganic phosphors; ion carriers, such as crown ether-attached or derived dyes; or combinations thereof.Specific examples of suitable luminophores include, but are not limited to, Pd(II) octaethylporphyrin; Pt(II)-octaethylporphyrin; Pd(II) tetraphenylporphyrin; Pt(II) tetraphenylporphyrin; Pd(II) meso-tetraphenylporphyrin tetrabenzoporphine; Pt(II) meso-tetraphenylmethylbenzoporphyrin; Pd(II) octaethylporphyrinone; Pt(II) octaethylporphyrinone; Pd(II) meso-tetrakis(pentafluorophenyl)porphyrin; Pt(II) meso-tetrakis(pentafluorophenyl)porphyrin; Ru(II) tris(4,7-diphenyl-1,10-phenanthrene) phenanthroline)(Ru(dpp)3); Ru(II) tris(1,10-phenanthroline)(Ru(phen)3), tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate(Ru(bpy)3); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; iridium(III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin); indium(III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin)-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas red; Rhodamine B; Rhodamine 6G; Sulforhodamine; m-cresol; Thymol blue; Xylenol blue; Cresol red; Chlorophenol blue; Bromocresol green; Bromocresol red; Bromothymol blue; Cy2; Cy3; Cy5; Cy5.5; Cy7; 4-nitrophenol; Alizarin; Phenolphthalein; o-Cresolphthalein; Chlorophenol red; Calcium magnesium reagent; Bromo-xylenol; Phenol red; Neutral red; Nitrazine; 3,4,5,6-tetrabromophenolphthalein; Congo red; Fluorescein; Eosin; 2',7'-dichlorofluorescein; 5(6)-carboxy-fluorescein fluorescein; carboxy naphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid; semi-naphthorhodafluor; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)dichloride; (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron; octaethylporphyrin platinum(II); dialkylcarbocyanine; dioctadecylepoxycarbocyanine; fluorenylmethoxyphosgene; 7-amino-4-methylcoumarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof.

[0117] In some examples, the detectable moiety can include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), naphthofluorescein (NF), or a derivative or combination thereof.

[0118] The detectable moiety can be attached to the cell penetrating peptide portion at an amino group, a carboxylate group, or a side chain of any amino acid of the cell penetrating peptide portion (eg, at an amino group, a carboxylate group, or a side chain of any amino acid in a CPP).

[0119] Treatment

[0120] The disclosed compounds may also comprise a therapeutic moiety. In some instances, the cargo moiety comprises the therapeutic moiety. The detectable moiety may be linked to the therapeutic moiety or the detectable moiety may also serve as the therapeutic moiety. The therapeutic moiety refers to a group that, when administered to a subject, will reduce one or more symptoms of a disease or condition.

[0121] Therapeutic moieties may comprise a variety of drugs, including antagonists (e.g., enzyme inhibitors) and agonists (e.g., transcription factors that result in increased expression of a desired gene product (although, as will be appreciated by those skilled in the art, antagonistic transcription factors may also be used)), are included. In addition, therapeutic moieties include those agents that are directly toxic and / or capable of inducing toxicity to healthy and / or unhealthy cells in the body. Furthermore, therapeutic moieties are capable of inducing and / or eliciting the immune system to fight potential pathogens.

[0122] The therapeutic moiety can, for example, include an anti-cancer agent, an anti-viral agent, an anti-microbial agent, an anti-inflammatory agent, an immunosuppressant, an anesthetic agent, or any combination thereof.

[0123] In some examples, the therapeutic moiety can be a tumor suppressor, a small molecule or peptide-based inhibitor, an enzyme for intracellular enzyme replacement therapy, an oligonucleotide.

[0124] The therapeutic moiety may comprise an anticancer agent. Exemplary anticancer agents include 13-cis retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretin, tretinoin), Alkaban-AQ, Alkeran, All-trans Retinoic Acid, Interferon-alpha, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic Trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Blenoxane usulfex), C225, leucovorin, Campath, Camptosar, camptothecin-11, Capecitabine, Carac, carboplatin, carmustine, carmustine thin tablets, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab, Chlorambucil, cisplatin, Citrovorum factor Factor), Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposome, Cytosar-U, Cytoxan, Dacarbazine, Actinomycin D, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, DaunoXome, Decadron,Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase L-asparaginase), Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin ozogamicin), Gemzar, Gleevec, Lupron, Lupron microspheres, Matulane, Maxidex, Mechlorethamine, Mechlorethamine hydrochloride, Medralone, Medrol, Megace, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Mechlorethamine, Novaldex, Novantrone,Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG -L-asparaginase, phenylalanine mustard, Platinol, Platinol-AQ, prednisolone, prednisone, Prelone, procarbazine, PROCRIT, Proleukin, Prolifeprospan20 with carmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alpha-2a), Rubex, erythromycin hydrochloride, Sandostatin (Sa ndostatin), Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys, Tioguanidine Purine, Thioguanine Tabloid, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine sulfate, Vincasar Pfs, vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel tablets, Glivec, GM-CSF, Goserelin,Granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, HydrocortAcetate, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocodone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-α, Ifosfamide, IL 2. IL-11, Imatinib mesylate, Imidazole carboxamide, Interferon α, Interferon α-2b (PEG conjugate), Interleukin 2, Interleukin 11, Intron A (Interferon α-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin, Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. The therapeutic moiety may also include a biological drug, such as, for example, an antibody.

[0125] In some examples, the therapeutic moiety can comprise an antiviral agent, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), and the like.

[0126] In some examples, the therapeutic moiety can include an antibacterial agent such as acedapsone; acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amikacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; sodium aminosalicylate; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apalcillin sodium; apramycin; aspartocin; astromicin sulfate sulfate; avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium; bacitracin hydrochloride; bacitracin; bacitracin methylenedisalicylate; bacitracin zinc; bambermycin; benzoylpas calcium; berythromycin; betamicin sulfate; biapenem; biniramycin; biphenamine hydrochloride; bispyrithione magsulfex; butikacin; butirosin sulfate; capreomycin sulfate; carbadox; carbenicillin disodium disodium; carbenicillindanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor; cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium;cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; cefonicid monosodium; cefoperazone sodium sodium); ceforanide; efotaximesodium; cefotetan; cefotetan disodium; cefotiamhydrochloride; cefoxitin; cefoxitin sodium; cefimidazole; cefimidazole sodium; cefpiramide; cefpiramide sodium; cefpiramide sulfate; cefpodoxime proxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium; ceftriaxone sodium sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; ceftriaxone sodium; cephalexin; cefalexin hydrochloride; cephaloglycin; cephaloridine; cephalothin sodium; cephapirin sodium; cephradine; cetocycline hydrochloride; cetophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphanilate; chlortetracycline hydrochloride bisulfate); chlortetracycline hydrochloride; cinoxacin;ciprofloxacin; ciprofloxacin hydrochloride; cirolemycin; clarithromycin; linafloxacin hydrochloride; clindamycin; clindamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillin sodium; clooxyquin; colistimethate sodium sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone; daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline; denofungin; diaveridine; dicloxacillin; dicloxacillin sodium; dihydrostreptomycin sulfate; pyridine; dirithromycin; doxycycline; doxycycline calcium; doxycycline phosphate complex; doxycycline hydrochloride; droxacin sodium; enoxacin; epicillin; epitetracycline hydrochloride hydrochloride); erythromycin; erythromycin acetate stearate; erythromycin estolate; erythromycin ethylsuccinate; erythromycin glucoheptonate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin; fluxacillin; fludalanine; flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin; furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid; gentamicin sulfate; gloximonam; gramicidin;haloprogin; hetacillin; hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole; isepamicin; isoniazid; josamycin; kanamycin sulfate sulfate; kitasamycin; levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef; mafenide; meclocycline; megalomicin potassium dihydrogen phosphate phosphate); mequidox; meropenem; methacycline; methacycline hydrochloride; methamphetamine; methamphetamine hippurate; methamphetamine mandelate; methicillin sodium; metioprim; metronidazole hydrochloride; metronidazole phosphate; mezlocillin; mezlocillin sodium; minocycline; minocycline hydrochloride; milinomycin hydrochloride; monensin; monensin sodium; nafcillin sodium; nalidixate sodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate; neomycin sulfate; neomycin undecylenate; netilmicin sulfate sulfate; neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone; nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole; nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium; ofloxacin;onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonam sodium; oxolinic acid; oxytetracycline; oxytetracycline calcium; oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin; pefloxacin; pefloxacin mesylate; penicillin G benzathine; penicillin G potassium; procaine penicillin G; penicillin G sodium; penicillin V; penicillin V benzathine; penicillin V hydrilamine; penicillin V potassium; pentizidone sodium; phenyl aminosalicylate; piperacillin sodium; piricicillin sodium; pirlimycin hydrochloride hydrochloride; pivampicillin hydrochloride; pivampicillin pamoate; pivampicillin probenate; polymyxin B sulfate; porfiromycin; propikacin; pyrazinamide; zinc pyrithione; quindecamine acetate; quinupristin; racephenicol; ramoplanin; ranimycin; relomycin; repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin; rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate; rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicin sodium phosphate; rosaramicin stearate; rosoxacin; roxarsone; roxithromycin; sancycline; sanfetrinemsodium; sarmoxicillin; sarpicillin; scopafungin;Sisomicin; Sisomicin sulfate; Sparfloxacin; Spectinomycin hydrochloride hydrochloride; spiramycin; stallimycin hydrochloride; steffimycin; streptomycin sulfate; streptonicozid; sulfabenz; sulfadiazine; sulfacetamide; sulfacetamide sodium; sulfacytine; sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene; sulfamerazine; sulfameter; sulfamethazine; sulfamethizole; sulfamethoxazole; sulfamonomethoxine; sulfamoxole; zinc sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet; sulfisoxazole; acetylsulfisoxazole; sulfisoxazole diethanolamine; sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillin hydrochloride hydrochloride); teicoplanin; temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprim; thiamphenicol; thiphencillin potassium; ticarcillin methylbenzyl sodium; ticarcillin disodium; ticarcillin monosodium; ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate; tosufloxacin;trimethoprim; trimethoprim sulfate; trisulfapyrimidine; troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin; vancomycin hydrochloride; virginiamycin; or zorbamycin.

[0127] In some instances, the therapeutic moiety can comprise an anti-inflammatory agent.

[0128] In some instances, the therapeutic moiety can comprise dexamethasone (Dex).

[0129] In other examples, the therapeutic moiety comprises a therapeutic protein. The protein can be genetically fused to the N-terminus or C-terminus of the MTD. Synthetic peptides containing non-natural amino acids can also be chemically conjugated to the side chains (e.g., unique cysteine) at the C-terminus of the MTD. Disclosed herein is the delivery of such enzymes / proteins to human cells by connecting enzymes / proteins to one of the disclosed cell-penetrating peptide motifs or MTD. The disclosed cell-penetrating peptide motifs have been tested with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C), and shown to work.

[0130] Targeting moiety

[0131] In some instances, the treatment moiety comprises a targeting moiety. The targeting moiety may comprise an amino acid sequence that may, for example, target one or more enzyme domains. In some instances, the targeting moiety may comprise an inhibitor for an enzyme that may work in a disease, such as cancer, cystic fibrosis, diabetes, obesity, or a combination thereof. For example, the targeting moiety may comprise any one of the sequences listed in Table 3.

[0132] Table 3. Exemplary targeting moieties

[0133]

[0134]

[0135]

[0136] *Fpa, Σ: L-4-fluorophenylalanine; Pip, Θ: L-homoproline; Nle, Ω: L-norleucine; Phg, Ψ: L-phenylglycine; F2Pmp, Λ: L-4-(phosphonodifluoromethyl)phenylalanine; Dap, L-2,3-diaminopropionic acid; Nal, Φ': L-β-naphthylalanine; Pp, θ: L-pipecolic acid; Sar, Ξ: sarcosine; Tm, trimesic acid.

[0137] The targeting moiety and the cell penetrating peptide moiety may overlap. That is, residues forming the cell penetrating peptide moiety may also be part of the sequence forming the targeting moiety, and vice versa.

[0138] The therapeutic moiety can be attached to the cell penetrating peptide portion at an amino group, a carboxylate group, or a side chain of any amino acid of the cell penetrating peptide portion (e.g., at an amino group, a carboxylate group, or a side chain of any amino acid of a CPP). In some examples, the therapeutic moiety can be attached to a detectable moiety.

[0139] In some examples, the therapeutic moiety can comprise a targeting moiety that can act as an inhibitor against Ras (eg, K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, etc., or a combination thereof.

[0140] Ras is a protein that in humans is encoded by the RAS gene. Normal Ras protein plays an important role in normal tissue signaling, and mutations in the Ras gene have been associated with the development of many cancers. Ras acts as a molecular on / off switch, and once it is turned on, Ras recruits and activates growth factors and other proteins necessary for the propagation of receptor signals. Mutated forms of Ras have been associated with a variety of cancers, including lung cancer, colon cancer, pancreatic cancer, and several leukemias.

[0141] Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical member of the PTP superfamily and plays many roles during eukaryotic cell signaling. PTP1B is a negative regulator of the insulin signaling pathway and is considered a promising potential therapeutic target, particularly for the treatment of type II diabetes. PIP1B has also been implicated in the development of breast cancer.

[0142] Pin1 is an enzyme that binds to a subset of proteins and plays a role in post-phosphorylation control in regulating protein function. Pin1 activity can modulate the outcome of proline-directed kinase signaling and, therefore, can regulate cell proliferation and cell survival. Dysregulation of Pin1 can play a role in a variety of diseases. Upregulation of Pin1 may be associated with certain cancers, while downregulation of Pin1 may be associated with Alzheimer's disease. Inhibitors of Pin1 may have therapeutic implications for cancer and immune disorders.

[0143] Grb2 is an adaptor protein involved in signal transduction and cell communication. The Grb2 protein contains an SH2 domain that binds to tyrosine phosphorylation sequences. Grb2 is widely expressed and is required for a variety of cellular functions. Inhibition of Grb2 function can impair developmental processes and block transformation and proliferation of various cell types.

[0144] Recently, it was reported that the activity of cystic fibrosis membrane conductance regulator (CFTR), a chloride channel protein mutated in cystic fibrosis (CF) patients, is negatively regulated by CFTR-associated ligand (CAL) through its PDZ domain (CAL-PDZ) (Wolde, M et al. J. Biol. Chem. 2007, 282, 8099). Inhibition of CFTR / CAL-PDZ interaction was shown to improve the activity of ΔPhe508-CFTR (the most common form of CFTR mutation) (Cheng, SH et al. Cell 1990, 63, 827; Kerem, BS et al. Science 1989, 245, 1073) by reducing its proteasome-mediated degradation (Cushing, PR et al. Angew. Chem. Int. Ed. 2010, 49, 9907). Therefore, disclosed herein are methods for treating subjects with cystic fibrosis by administering an effective amount of a compound or composition disclosed herein. The compound or composition administered to a subject may comprise a therapeutic moiety, which may comprise a targeting moiety, which may act as an inhibitor against CAL PDZ.

[0145] In some embodiments, the therapeutic moiety is a nucleic acid. In some embodiments, the nucleic acid is an antisense compound. In some embodiments, the antisense compound is selected from the group consisting of antisense oligonucleotides, small interfering RNA (siRNA), microRNA (miRNA), ribozymes, immunostimulatory nucleic acids, antagomir, antimir, microRNA mimics, supermir, Ul adapter and aptamer.

[0146] Also disclosed herein are compositions comprising the compounds described herein.

[0147] Also disclosed herein are pharmaceutically acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases according to specific substituents found on the compounds. Under conditions where the compounds disclosed herein have sufficient alkalinity or acidity to form stable non-toxic acid or base salts, it may be appropriate to administer the compounds in the form of salts. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-sugar phosphate, maleic acid, toluenesulfonic acid, methanesulfonic acid, etc. Thus, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propionates, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malonates, ascorbates, α-ketoglutarate, α-sugar phosphates, maleates, toluenesulfonates, and mesylates. Pharmaceutically acceptable salts of compounds can be obtained using standard methods well known in the art, for example, by reacting sufficiently basic compounds such as amines with suitable acids providing physiologically acceptable anions. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0148] Connectors

[0149] In various embodiments, the joint is covalently bound to the amino acid on the membrane translocation domain. The joint can be any part of two or more conjugated to the cargo portion in the membrane translocation domain. In some embodiments, the joint can be an amino acid. In other embodiments, the precursor of the joint can be any suitable molecule that can form two or more bonds with the amino acid in the membrane translocation domain and the cargo portion. Therefore, in various embodiments, the precursor of the joint has two or more functional groups, and each functional group can form a covalent bond with the membrane translocation domain and the cargo portion. For example, the joint can be covalently bound to the N-terminus, C-terminus or side chain of any amino acid in the membrane translocation domain or a combination thereof. In a specific embodiment, the joint forms a covalent bond between the membrane translocation domain and the cargo portion.

[0150] In some embodiments, the linker is selected from the group consisting of at least one amino acid, alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ether, each of which may be optionally substituted as defined above. For example, each of these linkers may be 1 to 500 atoms in length, such as 1 to 100, 1 to 250, 10 to 200, 25 to 300 atoms in length. Non-limiting examples of linkers include polyethylene glycol, optionally conjugated to lysine residues. In other examples, the linker may be a divalent or trivalent C1-C 50 Saturated or unsaturated straight or branched alkyl, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heteroaryl, cycloalkyl or cycloalkenyl.

[0151] In some embodiments, the linker is covalently bound to the N-terminus or C-terminus of an amino acid on a CPP motif, or to a side chain of glutamine, asparagine or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). In specific embodiments, the linker forms a bond with the side chain of glutamine on a CPP motif. In other specific embodiments, the linker described herein has the structure of L-1 or L-2:

[0152]

[0153] in

[0154] AA s It is the side chain or end of an amino acid on a peptide or main chain;

[0155] AA c is the side chain or end of the amino acid of cCPP;

[0156] p is an integer from 0 to 10; and

[0157] q is an integer from 1 to 50.

[0158] In other embodiments, the linker may be a proteolytically stable peptide sequence such as (GGS)n, (GGGS)n, (GSS)n, or (PAS)n, where n is 0-100.

[0159] In some embodiments, the linker is capable of releasing the cargo moiety from the membrane translocation domain after the polypeptide conjugate enters the cytoplasm of the cell. In some embodiments, the linker contains a group, or forms a group after binding to the membrane translocation domain and the cargo moiety, which is cleaved after cytoplasmic uptake of the polypeptide conjugate, thereby releasing the cargo moiety. Non-limiting examples of physiologically cleavable linking groups include carbonates, thiocarbonates, thioethers, thioesters, disulfides, sulfoxides, hydrazines, protease-cleavable dipeptide linkers, and the like.

[0160] For example, in an embodiment, the linker is covalently bound to the membrane translocation domain via a disulfide bond, such as a disulfide bond to the side chain of a cysteine ​​or cysteine ​​analog located in the membrane translocation domain or the cargo portion. In some embodiments, a disulfide bond is formed between a thiol group on a precursor of the linker and a side chain of a cysteine ​​or amino acid analog having a thiol group on a peptide, wherein the hydrogen bonds to each of the thiol groups are replaced by a sulfur atom bond. Non-limiting examples of amino acid analogs having thiol groups that can be used with the polypeptide conjugates disclosed herein are discussed above.

[0161] Preparation method

[0162] The compounds described herein can be prepared in a variety of ways known to those skilled in the art of organic synthesis or variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0163] The variation of compounds described herein includes the addition, subtraction or movement of various components as described for each compound. Similarly, when there are one or more chiral centers in a molecule, the chirality of the molecule can be changed. In addition, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by those skilled in the art. The chemical properties of protecting groups can be found in, for example, Wuts and Greene, Protective Groups in Organic Synthesis, 4th edition, Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0164] The starting materials and reagents used to prepare the disclosed compounds and compositions can be purchased from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or by methods known to those skilled in the art following the procedures described in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementary Editions (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, can be obtained from commercial sources.

[0165] The reactions to prepare the compounds described herein can be carried out in a solvent, which can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reactions are carried out. The reactions can be carried out in one solvent or in a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0166] The disclosed compounds can be prepared by expression and purification like any other protein. See Chen, K., & Pei, D. (2020). Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops. ACS chemical biology, 15 (9), 2568-2576, which is incorporated herein by reference for its teachings on protein preparation methods. Other methods for preparing the disclosed compositions involve solid phase peptide synthesis, in which the amino acid α-N-terminus is protected by a protective group that is sensitive to acid or base. Such protective groups should have stable properties for peptide connection formation conditions, and can be easily removed without destroying the growing peptide chain or racemizing any chiral center contained therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, tert-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-tert-butyloxycarbonyl, etc. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. For side chain amino groups such as lysine and arginine, other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and t-butyl, and for cysteine, triphenylmethyl (trityl). In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and are insoluble in the medium used. The solid support used for the synthesis of α-C-terminal carboxyl peptides is 4-hydroxymethylphenoxymethyl-co-poly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, which can be purchased from Applied Biosystems (Foster City, Calif.).The α-C-terminal amino acid is coupled to the resin by N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl) at temperatures between 10°C and 50°C in a solvent such as dichloromethane or DMF for about 1 to about 24 hours. When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine (preferably piperidine) prior to coupling with the α-C-terminal amino acid as described above. A method for coupling with a deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. The coupling of consecutive protected amino acids can be performed in an automatic peptide synthesizer. In one example, the α-N-terminus in the amino acids of the growing peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine (preferably piperidine). Each protected amino acid is then introduced at approximately 3-fold molar excess, and the coupling is preferably performed in DMF. The coupling agent can be O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously or in a single operation. The removal and deprotection of the polypeptide can be completed in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thioanisole, water, ethanedithiol and trifluoroacetic acid. In the case where the α-C-terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification (e.g., with methanol), followed by aminolysis or by direct transamidation. The protected peptide can be purified at this time or used directly in the next step. The removal of the side chain protecting groups can be accomplished using the above-mentioned cleavage mixture.The fully deprotected peptide can be purified by a series of chromatographic steps using any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography (e.g., on Sephadex G-25, LH-20) or countercurrent distribution; high performance liquid chromatography (HPLC), particularly reversed phase HPLC on octyl or octadecylsilyl-silica bonded phase column packings.

[0167] How to use

[0168] Also provided herein are methods of using the compounds or compositions described herein. Also provided herein are methods for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compounds or compositions described herein.

[0169] Also provided herein is a method for treating, preventing or improving cancer in a subject. The method includes administering to the subject an effective amount of one or more of the compounds or compositions described herein or their pharmaceutically acceptable salts. The compounds and compositions described herein or their pharmaceutically acceptable salts can be used to treat cancer in humans (e.g., children and elderly groups) and animals (e.g., veterinary applications). The disclosed method may optionally include identifying patients who need or may need to treat cancer. Examples of cancer types that can be treated by the compounds and compositions described herein include bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, urogenital cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer and testicular cancer. Other examples include anus, bile duct, bone, bone marrow, intestine (including colon and rectum), eye, gallbladder, kidney, mouth, larynx, esophagus, stomach, testicle, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells) cancer and / or tumors. Other examples of cancers that may be treated by the compounds and compositions described herein include carcinoma, Kaposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and others) and lymphomas (Hodgkin and non-Hodgkin), and multiple myeloma.

[0170] The methods for treating or preventing cancer described herein may further include treatment with one or more additional agents (e.g., anticancer agents or ionizing radiation). One or more additional agents as described herein and compounds and compositions or pharmaceutically acceptable salts thereof may be administered in any order, including simultaneous administration, and sequential administration with time intervals of up to several days. The method may also include more than a single administration of one or more additional agents and / or compounds and compositions or pharmaceutically acceptable salts thereof as described herein. The administration of one or more additional agents as described herein and compounds and compositions or pharmaceutically acceptable salts thereof may be by the same or different routes. When treated with one or more additional agents, compounds and compositions or pharmaceutically acceptable salts thereof as described herein may be combined into a pharmaceutical composition comprising one or more additional agents.

[0171] For example, a compound or composition as described herein, or a pharmaceutically acceptable salt thereof, can be combined with an additional anticancer agent into a pharmaceutical composition, such as 13-cis retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Fluorouracil, Agrylin, Ala-Cort, Aldesleukin, kin), alemtuzumab, alitretinoin, alkaban-AQ, melphalan, all-trans retinoic acid, interferon alpha, altretamine, amethopterin, amifostine, aminoglutethimide, anagrelide, anandron, anastrozole, cytarabine, abinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, arsenic trioxide, asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib , busulfan, Busulfex, C225, leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine Tablets, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine Liposomal, Cytosar-U, Cytoxan, Dacarbazine, Actinomycin D, Darbepoetin alfaalfa), Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposome, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa alfa), Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicinozogamicin), Gemzar, Gleevec, Lupron, Lupron Microspheres, Matulane, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Mechlorethamine, Nova ldex), Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, Phenylalanine mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan with carmustine implant20. Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alpha-2a), Rubex, Erythromycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide , Teniposide, TESPA, thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoramide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine sulfate, Vincasar Pfs, vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel tablets, Glivec, GM-CSF, Goserelin, granulocyte colony-stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocortisone acetate Acetate), hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocodone phosphate, hydroxyurea, ibritumomab, ibritumomab tiuxetan, idamycin, idarubicin, ifex, IFN-α, ifosfamide, IL2. IL-11, Imatinib mesylate, Imidazole carboxamide, Interferon α, Interferon α-2b (PEG conjugate), Interleukin 2, Interleukin 11, Intron A (Interferon α-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, LiquidPred, Lomustine, L-PAM, L-Sarcolysine Sin), Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase and LCR. Additional anticancer agents may also include biological drugs, such as, for example, antibodies.

[0172] Many tumors and cancers have viral genomes present in tumors or cancer cells. For example, Epstein-Barr Virus (EBV) is associated with many mammalian malignancies. The compounds disclosed herein can also be used alone or in combination with anticancer agents or antiviral agents such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treat patients infected with viruses that can cause cell transformation and / or to treat patients with tumors or cancers associated with the presence of viral genomes in cells. The compounds disclosed herein can also be used in combination with viral-based tumor disease treatments.

[0173] Also described herein is a method of killing tumor cells in a subject. The method includes contacting the tumor cells with an effective amount of a compound or composition as described herein, and optionally includes a step of irradiating the tumor cells with an effective amount of ionizing radiation. In addition, a method of tumor radiotherapy is provided herein. The method includes contacting the tumor cells with an effective amount of a compound or composition as described herein, and irradiating the tumor with an effective amount of ionizing radiation. As used herein, the term ionizing radiation refers to radiation containing particles or photons with sufficient energy or sufficient energy to produce ionization via nuclear interactions. An example of ionizing radiation is X-radiation. An effective amount of ionizing radiation refers to a dose of ionizing radiation that produces increased cell damage or death when administered in combination with a compound as described herein. Ionizing radiation can be delivered according to methods known in the art, including administration of radiolabeled antibodies and radioisotopes.

[0174] The methods and compounds described herein can be used for preventive and therapeutic treatments. As used herein, the term treatment (treating / treatment) includes prevention; delayed onset; alleviation, eradication or delay of worsening of signs or symptoms after onset; and prevention of recurrence. For preventive use, a therapeutically effective amount of a compound and composition as described herein or a pharmaceutically acceptable salt thereof is administered to a subject before the onset of cancer (e.g., before obvious signs of cancer), during early onset (e.g., after initial signs and symptoms of cancer), or after development is determined. Prophylactic administration may occur several days to years before symptoms of infection appear. Prophylactic administration can be used, for example, for chemopreventive treatment of subjects presenting precancerous lesions, subjects diagnosed with early malignancies, and subgroups susceptible to specific cancers (e.g., families, races, and / or occupations). Therapeutic treatment involves administering a therapeutically effective amount of a compound and composition as described herein or a pharmaceutically acceptable salt thereof to a subject after cancer is diagnosed.

[0175] In some examples of therapeutic methods for treating, preventing, or ameliorating cancer or tumors in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that may act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or a combination thereof.

[0176] The disclosed subject matter also relates to a method for treating a subject with a metabolic disorder or condition. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein are administered to a subject with a metabolic disorder and in need of treatment thereof. In some instances, the metabolic disorder may include type II diabetes. In some instances of methods of treating, preventing or improving a metabolic disorder in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety, which may act as an inhibitor for PTP1B. In a specific example of the method, the subject is obese, and the method may include treating the obesity of the subject by administering a composition as disclosed herein.

[0177] The disclosed subject matter also relates to methods for treating a subject with cystic fibrosis. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject with cystic fibrosis and in need of treatment thereof. In some examples of methods of treating cystic fibrosis in a subject, the compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety, which may act as an inhibitor against CAL PDZ.

[0178] Further disclosed are methods of using the disclosed compositions to deliver agricultural products to plant cells comprising contacting the cells with a peptide as disclosed herein. Compounds that can be delivered to plants include biodefense activators and biostimulants.

[0179] Compositions, formulations and methods of administration

[0180] Also disclosed herein are compositions comprising the compounds described herein.

[0181] Also disclosed herein are pharmaceutically acceptable salts and prodrugs of the disclosed compounds. Pharmaceutically acceptable salts include salts of the disclosed compounds prepared with acids or bases according to specific substituents found on the compounds. Under conditions where the compounds disclosed herein have sufficient alkalinity or acidity to form stable non-toxic acid or base salts, it may be appropriate to administer the compounds in the form of salts. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-sugar phosphate, maleic acid, toluenesulfonic acid, methanesulfonic acid, etc. Thus, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propionates, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malonates, ascorbates, α-ketoglutarate, α-sugar phosphates, maleates, toluenesulfonates, and mesylates. Pharmaceutically acceptable salts of compounds can be obtained using standard methods well known in the art, for example, by reacting sufficiently basic compounds such as amines with suitable acids providing physiologically acceptable anions. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0182] The in vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable method and technology currently or expectedly known to those skilled in the art. For example, the disclosed compounds can be formulated into physiologically or pharmaceutically acceptable forms and administered by any suitable route known in the art, including, for example, oral, nasal, rectal, topical and parenteral administration routes. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal and intrasternal administration, such as by injection. The administration of the disclosed compounds or compositions can be a single administration, or administered at continuous or different intervals, as can be easily determined by those skilled in the art.

[0183] The compounds disclosed herein and compositions comprising them can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform dosages over extended time periods. The compounds can also be administered in the form of their salt derivatives or in crystalline form.

[0184] The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources that are well known and easily available to those skilled in the art. For example, Remington's Pharmaceutical Science, EW Martin (1995) describes formulations that can be used in combination with the disclosed methods. In general, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with a suitable carrier to promote the effective administration of the compound. The composition used can also be in various forms. These forms include, for example, solid, semisolid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The composition also preferably contains conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents used with the compound include ethanol, dimethyl sulfoxide, glycerol, aluminum oxide, starch, saline, and equivalent carriers and diluents. To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain a total of between about 0.1 wt % to 100 wt % of one or more of the subject compounds, based on the weight of the total composition including carrier or diluent.

[0185] Formulations suitable for administration include, for example, sterile injection aqueous solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulation may be present in unit dose or multi-dose containers such as sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions, requiring only sterile liquid carriers (e.g., water for injection) before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, etc. It should be understood that, in addition to the ingredients specifically mentioned above, the compositions disclosed herein may also include other conventional agents in the art for the type of formulation discussed.

[0186] Compounds disclosed herein and compositions comprising them can be delivered to cells by direct contact with cells or via carrier means. Carrier means for delivering compounds and compositions to cells are known in the art, and include, for example, encapsulating the composition in a liposome portion. Another means for delivering compounds disclosed herein and compositions to cells includes attaching the compound to a protein or nucleic acid targeted for delivery to a target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow the composition to translocate across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transcellular membrane transport of biological parts for intracellular delivery. The compounds may also be incorporated into polymers, examples of which include poly(DL-lactide-co-glycolide) polymers for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] at a molar ratio of 20:80 (as used in GLIADEL); chondroitin; chitin; and chitosan.

[0187] For the treatment of tumor conditions, the compounds disclosed herein can be administered to patients in need of treatment in combination with other anti-tumor or anti-cancer substances and / or with radiation and / or photodynamic therapy and / or with surgical treatment to remove tumors. These other substances or treatments can be given simultaneously or at different times with the compounds disclosed herein. For example, the compounds disclosed herein can be used in combination with mitotic inhibitors (such as paclitaxel or vinblastine), alkylating agents (such as cyclophosphamide or ifosfamide), antimetabolites (such as 5-fluorouracil or hydroxyurea), DNA intercalators (such as doxorubicin or bleomycin), topoisomerase inhibitors (such as etoposide or camptothecin), anti-angiogenic agents (such as angiostatin), antiestrogens (such as tamoxifen) and / or other anti-cancer drugs or antibodies (such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.)) or immunotherapeutics (such as ipilimumab and bortezomib).

[0188] In certain instances, the compounds and compositions disclosed herein can be administered topically at one or more anatomical sites, such as sites of unwanted cell growth (such as tumor sites or benign skin growths, e.g., injection or topical application to tumors or skin growths), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. The compounds and compositions disclosed herein can be administered systemically, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier for oral delivery. They can be encapsulated in hard or soft shell gelatin capsules, can be compressed into tablets, or can be mixed directly with food in the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0189] The disclosed compositions are bioavailable and can be delivered orally. Oral compositions can be tablets, lozenges, pills, capsules, etc., and can also contain the following: binders, such as gum tragacanth, gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavoring agents such as peppermint, wintergreen oil, or cherry flavoring can be added. When the unit dosage form is a capsule, in addition to the above-mentioned types of materials, it can also contain a liquid carrier, such as a vegetable oil or polyethylene glycol. Various other materials can exist as coatings or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar, etc. Syrups or elixirs may contain the active compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavoring. Of course, any material used to prepare any unit dosage form should be pharmaceutically acceptable and substantially nontoxic in the amount employed. In addition, the active compound may be incorporated into sustained release formulations and devices.

[0190] The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly or intraperitoneally by infusion or injection. Solutions of the active agent or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof, and in oil. Under ordinary storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms.

[0191] The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder comprising an active ingredient, which is suitable for temporary preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under manufacturing and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium, including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using a surfactant. Optionally, the effect of microorganisms can be prevented by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar, buffer or sodium chloride. By including an agent that delays absorption, such as aluminum monostearate and gelatin, the extended absorption of injectable compositions can be achieved.

[0192] Sterile injectable solutions are prepared by incorporating the desired amount of the compounds and / or agents disclosed herein with the various other ingredients listed above in an appropriate solvent, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients present in the previously sterile-filtered solution.

[0193] For topical application, the compounds and agents disclosed herein can be applied as liquid or solid forms. However, it is generally desirable to apply them topically to the skin as a composition combined with a dermatologically acceptable carrier, which can be solid or liquid. The compounds and agents disclosed herein and compositions can be topically applied to the skin of a subject to reduce the size of malignant or benign growths (and may include complete removal), or to treat an infection site. The compounds and agents disclosed herein can be applied directly to the growth or infection site. Preferably, the compounds and agents are applied to the growth or infection site with formulations such as ointments, creams, lotions, solutions, tinctures, etc.

[0194] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silicon dioxide, alumina, and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the compound can be dissolved or dispersed at effective levels, optionally with the aid of nontoxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents may be added to optimize the properties for a given use. The resulting liquid composition may be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using, for example, a pump-type or aerosol sprayer.

[0195] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with the liquid carrier to form spreadable pastes, gels, ointments, soaps, etc., for application directly to the user's skin.

[0196] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.

[0197] The dosage range of the composition administration is a dosage range large enough to produce the desired effect affecting the symptom or condition. The dosage should not be large enough to cause adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Usually, the dosage will vary with the patient's age, condition, sex, and degree of disease, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by individual physicians. The dosage can vary and can be administered once or multiple times a day for one or more days.

[0198] Also disclosed are pharmaceutical compositions comprising a combination of a compound disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions suitable for oral, topical or parenteral administration comprising a certain amount of compound constitute preferred aspects. The dosage applied to a patient (particularly a human) should be sufficient to achieve a therapeutic response in the patient within a reasonable time frame without lethal toxicity, and preferably cause no more than an acceptable level of side effects or morbidity. Those skilled in the art will recognize that dosage will depend on a variety of factors, including the subject's condition (health), the subject's weight, the type of simultaneous treatment (if any), the frequency of treatment, the severity and stage of the treatment ratio and the pathological condition.

[0199] Also disclosed are kits containing compounds disclosed herein in one or more containers. The disclosed kits may optionally include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other components, adjuvants or auxiliary agents as described herein. In another embodiment, the kit includes one or more anticancer agents, such as those described herein. In one embodiment, the kit includes instructions or packaging materials describing how to administer the compounds or compositions of the kit. The container of the kit may have any suitable material, such as glass, plastic, metal, etc., and may have any suitable size, shape or configuration. In one embodiment, the compounds and / or agents disclosed herein may be provided in the kit as solids (such as tablets, pills or powder forms). In another embodiment, the compounds and / or agents disclosed herein may be provided in the kit as liquids or solutions. In one embodiment, the kit includes ampoules or syringes containing compounds and / or agents disclosed herein in liquid or solution form.

[0200] Several embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are within the scope of the following claims.

[0201] Examples

[0202] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and these examples are intended to be purely exemplary of the present invention and are not intended to limit the scope of what the inventors regard as their invention. However, those skilled in the art will appreciate from this disclosure that many changes may be made to the specific embodiments disclosed and still achieve the same or similar results without departing from the spirit and scope of the present invention.

[0203] Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

[0204] The human type III fibronectin (FN3) domain was selected as a scaffold for the membrane translocation domain (MTD) (Koide, A., et al. (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol. 284 (4): 1141-1151). FN3 is a small (90-100aa) highly stable protein and has been widely used to develop monomeric antibodies that bind to target proteins with high affinity and specificity (Chandler, PG, et al. (2020) Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells 9 (3): 610). Previous studies have shown that several loop regions of FN3 can tolerate mutations (Steven, A., et al. (2012) Design of novel FN3 domains with high stability by a consensus sequence approach, Protein Engineering, Design and Selection, 25 (3): 107-117). Additionally, FN3 does not contain any cysteine ​​or disulfide bonds and is therefore stable in the intracellular environment. FN3 can be easily folded into its native form without any physical or chemical assistance and can be produced in high yields in Escherichia coli. Finally, FN3 is derived from an abundant human extracellular protein and is unlikely to trigger any immune response.

[0205] Design, expression and purification of MTD.

[0206] The BC, DE, and FG loops of FN3 have previously been shown to be highly tolerant to sequence mutations. The GDSPAS sequence of the FG loop was replaced with RRRWWW (SEQ.ID.NO.: 104) to obtain MTD1 (Table 4). Together with the existing arginine residues in the FG loop, this generates a putative CPP motif (R4W3) without changing the loop size. Similarly, the tetrapeptide AVTV of the BC loop was replaced with WWWRRR (SEQ.ID.NO.: 105) to utilize the existing arginine in the loop to form a putative CPP (W3R4) (Table 4). The size of the BC loop in the resulting mutant MTD2 increased by 2 residues. In order to explore the possibility of transplanting the CPP motif to the other end of FN3, the tripeptide NSP in the CD loop was replaced with the CPP motif R4W3 to obtain MTD3. To test the feasibility of transplanting CPP sequences into two different loops, the relatively hydrophobic tripeptide (VTV) in the BC loop was replaced with WYW, and the hydrophilic motif (GDSPAS; SEQ.ID.NO.: 106) in the FG loop was replaced with RRR to produce MTD4. Finally, MTD5 was generated by exchanging the WYW and RRR motifs of MTD4. Two mutants, MTD4a and MTD4b, were also generated, which contained only half of the CPP motif in the BC loop and the FG loop, respectively, to test the relative importance of the RRRR and WYW motifs. The WYW motif is more hydrophilic than WWW and has previously been reported as an "endosome escape motif" for cell-permeable antibodies (Kim, J.-S., et al. (2016) Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomalescape. J. Control. Rel. 235: 165-175). The loop insertion mutants were analyzed by the online program Phyre2 to predict their folded structures based on sequence homology. All mutants maintained an overall fold similar to wild-type FN3, with the CPP motif displayed on their surface and constrained to a "loop-like" topology ( Figure 1 ).

[0207] To further improve the properties of MTD4 (e.g., cell entry efficiency, metabolic stability, and expression yield), the BC and FG loops of FN3 were replaced with different combinations of Y, W, A, and R residues to generate MTD6-10 (Table 4). The overall cell entry efficiency of MTD6-10 was assessed by labeling MTD at a unique C-terminal cysteine ​​with tetramethylrhodamine-5-maleimide (TMR). HeLa cells were treated with TMR-labeled proteins (5 μM) for 2 hours and analyzed by live cell confocal microscopy. MTD7 TMR and MTD9TMR Shown to be related to MTD4 TMR Similar uptake, while MTD6 TMR 、MTD8 TMR and MTD10 TMR Show less cell entry (Fig. 3 A to Fig. 3 I).In addition, the separation yield of MTD6-10 changes between the Escherichia coli cell culture of 0.6 to 6.2mg / L (Table 4).It should be noted that MTD4 and MTD6 are only slightly different in BC loop sequence ("WYW" to "YWW"), but there are significant differences in separation yield (9.4mg / L to 0.9mg / L) and cell entry efficiency.Similarly, exchanging CPP motifs between the BC loop and FG loop of MTD4 can cause poor expression and variants (MTD5 in Table 4) with much lower activity.These results prove that the correct folding / stability of MTD and high cell entry efficiency not only require the presence of amphipathic CPP motifs, but also require their correct presentation on protein surface.

[0208] Table 4. Loop sequences and expression yields of MTD

[0209]

[0210] *During mutagenesis, underlined residues were deleted and bold residues were inserted.

[0211] The DNA sequence encoding WT FN3 was chemically synthesized and ligated to the prokaryotic expression vector pET-15b. To facilitate protein purification and genetic fusion with cargo proteins, a hexa-histidine tag and a thrombin cleavage site were added to the N-terminus of FN3, along with a flexible linker sequence (GGSGGSGGS; SEQ.ID.NO.: 107), followed by a recognition site for the restriction endonuclease Sad, and a cysteine ​​was added to its C-terminus (Table 5). All loop insertion mutants were generated by a one-step polymerase chain reaction (PCR) method (Qi, D., et al. (2008) A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. J. Virol. Meth. 149: 85-9020) and expressed in E. coli. Among the mutant proteins, MTD1 failed to produce a significant amount of soluble protein, while WT FN3 and MTD2-5 produced good yields of soluble protein (Table 4). FIG. 2A to FIG. 2B Expression and purification of MTD4 is shown as an example. All proteins were purified to near homogeneity by metal affinity chromatography on a Ni-NTA column.

[0212] Cloning, expression and purification of MTD.

[0213] All loop insertion mutants were generated by a one-step polymerase chain reaction (PCR) method (Qi, D., supra). The peptide sequence of each construct was confirmed by sequencing the entire coding region of the plasmid DNA (Table 5). Pilot-scale protein expression was performed to check the expression level of the mutant proteins, and all mutants were expressed in 5 mL E. coli BL21 (DE3) bacterial culture. Induction was performed at 37°C in the presence of 0.25 mM IPTG. Expression levels were checked by comparing total cell lysates before and after induction on SDS gels ( FIG. 2A to FIG. 2B ).

[0214] Table 5. Amino acid sequences of WT FN3, MTD and MTD-cargo fusions

[0215]

[0216]

[0217] Large-scale expression conditions are the same as those of small-scale expression conditions, and Escherichia coli cells are centrifuged and stored at -80 ° C. Use lysis buffer (50 mL of wash buffer, 0.2 mg / mL lysozyme, 2 mM β-mercaptoethanol, 2 mM PMSF, 2 Roche complete protease inhibitor mixtures) to lyse these cells. After the cell plate is evenly resuspended in the lysis buffer, the cells are ultrasonically treated twice (Amp.70%). The crude cell lysate is centrifuged (12000 g for 20 minutes) and soluble cell lysate is collected. Protein purification is carried out using fast protein liquid chromatography (FPLC), and soluble cell lysate is loaded onto a Ni-NTA column (with 15 mM imidazole). The column is thoroughly washed with wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5% glycerol and 50 mM imidazole). The wash buffer containing a linear gradient of 50-500 mM imidazole (pH 7.4) is used to elute the protein in 30 minutes.

[0218] Cloning, expression and purification of MTD4-PTP1B, MTD4-NS1, MTD4-RBDV, MTD4-SEP, MTD4-GFP11 and MENC.

[0219] Use plasmid DNA as template and use the primer containing SacI and BamHI restriction site at 5 ' and 3 ' end of PTP1B coding sequence respectively, by PCR amplification PTP1B coding sequence (amino acid 1-321).PCR product is digested with restriction enzyme SacI and BamHI and joined to plasmid pET-15b-MTD4 with identical two kinds of enzyme linearization.This causes PTP1B to merge with the C-terminal of MTD4.Other plasmids based on pET15b of coding MTD4-RBDV, MTD4-NS1, MTD4-SEP and MENC fusion protein are also constructed in a similar manner, just add XhoI restriction site instead of BamHI at 3 ' end of RBDV, NS1, SEP and ENC coding sequence. The GFP11 peptide was inserted into the C-terminus of MTD4 by a one-step PCR reaction (D. Qi, et al., "A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis," J Virol Methods, 149(1):85-90, 2008). The authenticity of the DNA construct was confirmed by restriction mapping and sequencing of the entire coding sequence.

[0220] E. coli BL21 (DE3) cells transformed with the appropriate plasmids were grown in LB medium supplemented with 75 mg / L ampicillin at 37°C. 600When reaching 0.6, cells were induced by adding 0.25mM IPTG for 4 hours at 37°C. Cells were precipitated by centrifugation. For MTD4-RBDV, the cell pellet was resuspended in 50mL (per liter of cell culture) lysis buffer [50mM Tris (pH 7.4), 150mM NaCl, 25mM imidazole, 3mM β-mercaptoethanol, protease inhibitor cocktail tablets and 20mg / ml lysozyme]. The cells were briefly sonicated and centrifuged at 12,000g for 20 minutes. The crude lysate was loaded onto a 5-mL Histrap Ni-NTA column attached to FPLC. The column was thoroughly washed with wash buffer (50mM Tris, pH 7.4, 300mM NaCl, 5% glycerol and 50mM imidazole). Protein was eluted with a wash buffer containing 500mM imidazole. MTD4-PTP1B, MTD4-SEP, MENC, MTD4-GFP11 and MTD4-NS1 were purified in a similar manner, but for MTD4-NS1, the cell pellet was resuspended in 50 mM Tris (pH 8), 500 mM NaCl, 25 mM imidazole, 3 mM β-mercaptoethanol, protease inhibitor cocktail tablets and 20 mg / ml lysozyme. MENC protein was expressed in BL21 Rosetta pLysS cell line and induced overnight at 18°C. The specific activity of MTD4-PTP1B was determined using p-nitrophenyl phosphate (pNPP) as a substrate, and the specific activity of MTD4-PTP1B was found to be similar to that of WT PTP1B.

[0221] Cell entry efficiency of MTD.

[0222] Wild-type FN3 and MTD were fluorescently labeled with tetramethylrhodamine-5-maleimide (TMR) at their single C-terminal cysteine. HeLa (human cervical carcinoma) cells were incubated with TMR-labeled proteins and imaged by confocal microscopy without fixation. Interestingly, WT FN3 showed significant cellular entry, although the intracellular fluorescence pattern was punctate, suggesting that most of the internalized protein was trapped within endosomes / lysosomes (Figure 3A). In contrast, in addition to the punctate fluorescence, MTD4 TMR Treated HeLa cells also exhibited clearly visible diffuse fluorescence throughout the cell volume (including the nucleus) (Figure 3B). The presence of diffuse intracellular fluorescence indicates that internalized MTD4 TMR A large fraction of MTD2 has left the endosome and successfully reached the cytoplasm (and nucleus). TMR The fluorescence pattern of the treated cells was intermediate between that of FN3- and MTD4-treated cells; although some diffuse fluorescence was visible, the fluorescence was mainly punctate (Fig. 3B). TMRNo significant intracellular fluorescence was produced (data not shown).

[0223] To quantify the efficiency of cell entry, cells were next analyzed by flow cytometry and the results were compared with those of CPP12, a previously reported efficient cyclic CPP. Preliminary data after adjusting for the degree of dye labeling of the proteins indicated that MTD4 entered HeLa cells more efficiently than FN3 or MTD2, but less efficiently than CPP12 ( Figure 4 ).

[0224] Intracellular delivery of PTP1B.

[0225] In order to prove that MTD functionally delivers protein cargo to the cytoplasm of mammalian cells, protein tyrosine phosphatase 1B (PTP1B) is selected as cargo, and its gene is fused to the C-terminus of MTD4. Tyrosyl phosphorylation is usually confined to the cytoplasmic domain of cytoplasm and nucleoprotein or transmembrane protein. PTP1B is a kind of broad specificity phosphatase, which can catalyze the dephosphorylation (Selner, NG, et al. (2014) Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosinephosphatases.Biochem.53 (2): 397-412) of many intracellular proteins. The cytoplasmic delivery of PTP1B is expected to reduce the phosphotyrosine (pY) level of intracellular protein, which can be easily monitored by anti-pY protein blotting. NIH3T3 cells are treated with different concentrations of MTD4-PTP1B for 4 hours, washed, and cracked in the presence of protease and phosphatase inhibitors. Cellular proteins are separated by SDS-PAGE, transferred to nitrocellulose membrane, and blotted with anti-pY antibody 4G10. MTD4-PTP1B caused a dose-dependent decrease in pY levels in HeLa cells, and at 5 μM pY was almost completely lost ( Figure 5 ).

[0226] Intracellular delivery of Ras inhibitors.

[0227] To further demonstrate the utility of MTD4 and generate a cell-permeable protein with therapeutic potential, MTD4 was fused to two previously reported proteins that bind to mutant KRas with high affinity and specificity. Ras mutations (including KRas, HRas, and NRas) are found in about 30% of human cancers, making Ras one of the most important cancer drug targets (Prior, IA, et al. (2012) A comprehensive survey of Ras mutations in cancer. Cancer Res. 72 (10): 2457-67; Khan, I., et al. (2020) Therapeutic targeting of RAS: New hope for drugging the "undruggable. Biochim. Biophys. Acta, Mol. Cell Res. 1867, 118570). Unfortunately, Ras is one of the most challenging (and undruggable) drug targets because it is located inside the cell and has no major binding pocket on its surface for small molecules to bind to. To date, several small molecules that covalently modify (and inhibit) G12C mutant KRas have entered the clinic, and one of them has been approved by the FDA, validating Ras as a viable target for treating Ras mutant cancers (Moore, AR, et al. (2020) RAS-targeted therapies: is the undruggable drugged? Nature Rev. Drug Disc. 19(8): 533-552). Recently, a potent non-covalent inhibitor that is selective for KRas G12D mutants has also been reported (Wang, X., et al. (2021) Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12DInhibitor. J.Med.Chem.10.1021 / acs.jmedchem.1c01688). However, many other Ras mutants (e.g., G12V, G12S, G13C, and Q61H) do not have such an inhibitor.

[0228] Previous researchers have generated potent KRas protein inhibitors through phage display screening. Wiechmann et al. screened a phage display library of C-Raf Ras-binding domain (RBD) and identified several variant RBDVs that bound to the effector binding site of GTP-bound HRas in the same manner as the WT C-Raf RBD (K DAbout 3nM) with about 20 times higher affinity binding (Wiechmann, S., et al. (2020) Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids. J. Biol. Chem. 295 (14): 4526-4540). Expression of RBDV in Ras mutant cancer cells blocks Ras signaling and causes cancer cell apoptosis. Similarly, Koide and colleagues screened a large phage-displayed FN3 mutant library for HRas and found an effective binder, called "NS1", which binds to the allosteric site and prevents Ras from dimerizing on the plasma membrane (Spencer-Smith, R., et al. (2017) Inhibition of RAS function through targeting an allosteric regulatory site. Nat. Chem. Biol. 13: 62-68). When expressed intracellularly, NS1 also inhibits Ras signaling and induces apoptosis in Ras mutant cancer cells (Khan, I., et al. (2019) Targeting the α4-α5 dimerization interface of K-RAS inhibits tumor formation in vivo. Oncogene 38(16): 2984-2993). Unlike RBDV (which binds to all Ras isoforms (KRas, HRas, and NRas) in the GTP-bound state), NS1 binds to HRas (K D =13 nM) and KRAS (K D =65 nM) is specific and independent of the nucleotide identity. Unfortunately, RBDV and NS-1 are not suitable as therapeutic agents because they cannot penetrate the cell membrane to reach the target protein.

[0229] One of the RBDVs (RBDV3) or NS1 genes was fused to the C-terminus of MTD4 to generate MTD4-RBDV and MTD4-NS1, respectively. The proteins were expressed in Escherichia coli and purified to near homogeneity by metal affinity chromatography. The fusion proteins were next tested for their ability to reduce the viability of Ras mutant cancer cells. MTD4-RBDV dose-dependently reduced the viability of non-small cell lung cancer cells (H358), pancreatic ductal adenocarcinoma cells (Mia PaCa-2), non-small cell lung cancer cells (A549), and colorectal cancer cells (SW480), with IC 50The values ​​were 1.2±0.2, 1.2±0.1, 1.7±0.2 and 2.2±0.4 μM, respectively ( FIG6A ). Similarly, MTD4-NS1 dose-dependently reduced the viability of the above cells, with IC 50 The values ​​were 1.4 ± 0.2, 1.5 ± 0.1, 1.8 ± 0.1 and 0.9 ± 0.2 μM, respectively ( FIG6B ). To verify the selectivity of NS1 for KRas and HRas but not NRas, H1915 non-small cell lung cancer cells (HRAS Q61L ) mutants and H1299 non-small cell lung cancer cells (NRAS Q61K ) mutants. MTD4-RBDV showed activity in both cells, where IC 50 4.7±1.1μM and IC 50 Surprisingly, MTD4-NS1 showed activity in both cell types, with an IC 50 1.5±0.2μM and IC 50 NS1 was fused to the N-terminus of MTD4 to generate NS1-MTD4. NS1-MTD4 also dose-dependently reduced the viability of H358 and MiaPaCa-2, with IC 50 1.1±0.1μM and IC 50 1.8±0.2μM( Figure 6C NS1-MTD4 also reduced the viability of H1299 cells, with IC 50 It is 1.6±0.2μM.

[0230] To determine whether the loss of cancer cell viability was caused by on-target inhibition of Ras effector protein interactions and Ras signaling, a bioluminescence resonance energy transfer (BRET) assay developed by Rabbitts and colleagues was used (Bery, N., et al. (2018) BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions. eLife 7: e37122). Human embryonic kidney (HEK293T) cells that do not carry any Ras mutations and are relatively insensitive to Ras inhibitors were transfected with plasmid DNA encoding KRas G12V (or G12D)-luciferase and c-Raf RBD-green fluorescent protein (GFP) fusions. Upon addition of the membrane-permeable luciferase substrate coelenterazine 400a, the interaction between the KRas mutants and the RBD generates a BRET signal from the luciferase donor to the GFP acceptor. Inhibitors that block the Ras-RBD interaction are expected to reduce the BRET signal. Figure 7 As shown in Figure , MTD4-RBDV dose-dependently reduced the BRET signal in HEK293T cells transfected with KRas G12V or G12D mutants, where IC 50 Values ​​were 5-10μM. MTD4-NS1 also reduced the BRET signal, but not as effectively as MTD4-RBDV, which is very consistent with their relative Ras binding affinities. In addition, unlike RBDV, NS1 does not bind to the effector binding site and does not directly compete with Raf for binding to HRas or KRas. Instead, NS1 binds to the dimerization site of HRas / KRas and indirectly inhibits the Ras-Raf interaction. It should be noted that for MTD4-NS1, the BRET ratio drops suddenly at 10μM protein; this may be due to the partial loss of HEK293T cell viability at high inhibitor concentrations. The latter again suggests that in addition to on-target inhibition of Ras signaling, MTD4-NS1 may also have off-target effects.

[0231] The on-target activity of the fusion protein was further assessed by examining the phosphorylation levels of Ras downstream signaling proteins by Western blot analysis. Ras activates the Raf / MEK / ERK and PI3K / Akt signaling pathways and increases the phosphorylation of protein kinases MEK, ERK, and Akt. Inhibition of Ras function reduces the phosphorylation levels of Akt and MEK. In fact, treatment of MiaPaCa-2 cells with MTD4-RBDV dose-dependently reduced the phosphorylation of Akt and MEK, with IC 50The values ​​were 1-3 μM, while total Akt and MEK levels remained relatively constant (Figure 8A). MTD4-NS1 also reduced p-Akt and p-MEK levels, but not as effectively as MTD4-RBDV (Figure 8B).

[0232] We tested whether MTD4-RBDV and MTD4-NS1 induce apoptosis in Ras mutant cancer cells. Therefore, H358 lung cancer cells were treated with the fusion proteins for 24 h and stained with Alexa Fluor TM 488-Annexin V and propidium iodide staining. MTD4-RBDV showed a dose-dependent increase in the Annexin V-positive cell population, indicating that apoptosis was responsible for the loss of viability observed in the Cell Glo assay ( Fig. 9 MTD4-NS1 also induces strong apoptosis at a concentration as low as 2.5 μM ( Fig. 9 ).

[0233] Cytoplasmic delivery efficiency of MTD4.

[0234] Among the 10 MTDs generated, MTD4 has a relatively high expression yield in Escherichia coli, and has an excellent total cell entry efficiency as monitored by confocal microscopy. Therefore, MTD4 is selected for further evaluation, and its cytoplasmic delivery efficiency is determined using two different methods. First, green fluorescent protein (GFP) complementation assay is used, wherein the combination of 16-aa peptide GFP11 (corresponding to the 11th β chain of GFP) and superfolded GFP1-10 is involved to form functional GFP. GFP11 gene is fused to the C-terminal of MTD4. As a comparison, GFP11 is also chemically conjugated with CPP12. HEK293T cells are transiently transfected to express GFP1-10 protein, incubated with 10 μM GFP11, MTD4-GFP11 or CPP12-GFP11 for 6 hours, and examined by live cell confocal microscopy. Cells treated with MTD4-GFP11 show strong and diffuse fluorescence (Figure 10A to Figure 10D) in the entire cell volume. Cells treated with CPP12-GFP11 also showed strong fluorescence, although the signal was more punctate. In contrast, untreated cells and cells treated with unconjugated GFP11 showed little fluorescence.

[0235] HiBit, an 11-residue peptide derived from NanoLuc (VSGWRLFKKIS) (SEQ ID NO: 151), was conjugated to FN3, MTD, and CPP12 via disulfide bonds using a previously reported luciferase complementation assay (SLYTeo, J et al., "Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay," Nat Commun, 12(1): 3721, 2021). HEK293T cells were transfected with plasmid DNA encoding the 18-kDa subunit of NanoLuc (LgBit) and then incubated with HiBit or HiBit conjugates. After successful delivery to the cytoplasm, the HiBit peptide is released from the conjugate via intracellular thiols (e.g., glutathione) and specifically binds to the cytoplasmic LgBit to form catalytically active luciferase, whose activity can be quantified in real time by adding the cell permeable substrate furimazine. CPP12-SS-HiBit, MTD4-SS-HiBit, and MTD2-SS-HiBit increased the luciferase activity of HEK293T cells in a dose-dependent manner, whereas FN3-SS-HiBit did not ( Fig.11 ). Interestingly, MTD4 and CPP12 showed similar cytoplasmic delivery efficiency at high concentrations (e.g., 5 μM); however, at lower concentrations (e.g., 0.19 and 0.56 μM), MTD4 was 5-10 times more active than CPP12 (Figure 10A to Figure 10D). Unconjugated HiBit also produced some luciferase activity at high concentrations, probably because HiBit has weak intrinsic cell penetration activity (given its amphipathic sequence) (MK Schwinn et al., "CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide," ACS Chem Biol, 13 (2): 467-474, 2018).

[0236] Intracellular delivery of additional protein cargo.

[0237] MTD4 first tested its ability to deliver protein cargo Superecliptic pHluorin (SEP). SEP is a pH-sensitive variant of GFP (pKa about 7.2); it has high fluorescence in the neutral environment (pH 7.4) of mammalian cytoplasm, but is essentially non-fluorescent in endosomes (pH 5.5-6.5) or lysosomes (pH 4.5-5.5) (S. Sankaranarayanan, et al., "The use of pHluorins for optical measurements of presynaptic activity," Biophys J, 79 (4): 2199-208, 2000). Therefore, any intracellular fluorescence largely reflects the amount of SEP that has successfully reached the cytoplasm. The SEP gene was fused to the C-terminus of MTD4, and the MTD4-SEP fusion protein was purified from Escherichia coli. HeLa cells were incubated with 5 μM SEP or MTD4-SEP for 2 hours and imaged by live cell confocal microscopy. Cells treated with MTD4-SEP showed strong fluorescence throughout the cell volume, whereas untreated cells or cells treated with SEP had no detectable fluorescence ( FIGS. 12A to 12C ).

[0238] In order to prove that protein cargo is functionally delivered to the cytoplasm of mammalian cells, protein tyrosine phosphatase 1B (PTP1B) is selected as cargo, and its gene is fused to the C-terminus of MTD4. Tyrosyl phosphorylation is usually confined to the cytoplasmic domain of cytoplasm and nucleoprotein or transmembrane protein. PTP1B is a broad specificity phosphatase, which can catalyze the dephosphorylation of many intracellular proteins (NGSelner et al., "Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases," Biochemistry, 53 (2): 397-412, 2014). The cytoplasmic delivery of PTP1B is expected to reduce the phosphotyrosine (pY) level of intracellular protein, which can be easily monitored by anti-pY protein blotting. HEK293T cells are treated with different concentrations of MTD4-PTP1B for 6 hours, washed, and cracked in the presence of protease and phosphatase inhibitors. Cellular proteins are separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-pY antibody 4G10. MTD4-PTP1B dose-dependently reduced the overall pY level in HEK293T cells, where EC 50Values ​​≤ 5 nM ( FIGS. 13A-13B ). Based on the fact that cells treated with 5 nM MTD4-PTP1B contained lower pY levels than cells treated with 5 μM unconjugated PTP1B (WT), it was estimated that MTD4 increases the cytoplasmic entry of PTP1B by >1000-fold.

[0239] In vivo biodistribution of MTD4-EGFP-NLS-Cre.

[0240] In order to study the biodistribution of MTD4 in mice, the EGFP-NLS-Cre protein gene was fused to the C-terminus of MTD4. Cre recombinase is an enzyme derived from P1 bacteriophage and can catalyze site-specific DNA recombination (K. Abremski, et al., "Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein," J Biol Chem, 259 (3): 1509-14, 1984). MTD4-EGFP-NLS-Cre (MENC) fusion protein was successfully delivered to cells of transgenic mice, and it is expected to cause DNA recombination events, thereby activating the expression of red fluorescent protein (mCherry). Primary cells derived from transgenic mice were treated in vitro with 1 μM MENC, resulting in mild expression of mCherry after 48 hours (data not shown).

[0241] Inspired by the in vitro data, MENC (8 mg / kg) was injected into the tail vein of four transgenic mice; two mice were euthanized 3 h after treatment, while the others were euthanized 48 h after treatment. Different organs were harvested, fixed, embedded, and sectioned for observation under confocal microscopy. Samples were treated with a pure black dye to reduce tissue autofluorescence. Strong EGFP fluorescence was observed in most tissues except the brain, indicating that MENC is widely biodistributed into different organs ( Fig.14 On the other hand, the mCherry signal in most organs was still weak even after 48 h, which may be due to the fact that the nuclear localization sequence (NLS) is flanked by two protein domains and the nuclear localization efficiency is poor. Nevertheless, the confocal images clearly demonstrated the biodistribution of MENC in different mouse organs.

[0242] Serum stability of MTD4.

[0243] MTD4 was incubated with human serum for different time periods and analyzed by SDS-PAGE. MTD4 appears to be proteolytically cleaved at a single site near the C-terminus, as the cleavage product is only slightly smaller than MTD4 and is able to bind to a nickel affinity column via its N-terminal 6xHis tag ( FIG. 15A ). 1 / 2 The cleavage was about 3 hours and was essentially complete after 24 hours. In comparison, FN3 appeared to undergo similar cleavage, but t 1 / 2 >24 hours (Figure 15B). Mass spectrometry analysis confirmed the cleavage of FN3 and MTD4.

[0244] Other suitable MTD brackets.

[0245] Although the current MTDs are all derived from the 10th FN3 domain of human fibronectin, it is hypothesized that many other protein domains may also serve as appropriate scaffolds to engineer additional MTDs. In general, a good scaffold should contain two or more adjacent surface loops (for incorporation of two CPP motifs), be free of disulfide bonds and stably folded (to tolerate sequence variation), be expressed in high yields in E. coli or eukaryotic hosts, and have no biological function itself. Based on these criteria, examples of suitable MTD scaffolds include other FN3 domains of fibronectin (Kornblihtt AR, et al., Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 4(7): 1755-1759, 1985), nanobodies (Muyldermans S., "Nanobodies: natural single-domain antibodies." Annu Rev Biochem. 82: 775-797, 2013), designed ankyrin repeat proteins (DARPins) (Stumpp MT, et al., "DARPins: a true alternative to antibodies." Curr Opin Drug Discov Devel. 10(2): 153-159, 2007), consensus triangular tetrapeptide repeats (CTPRs) (Uribe KB, et al., "Engineered Repeat Protein Hybrids: The New Horizon for Biologic Medicines and Diagnostic Tools." Acc Chem Res.54(22):4166-4177,2021)、Anticalin (Rothe C, et al., Anticalin TMBioconjug Chem.28(9):2361-2371,2017), Affimer (Tiede C, et al., "Affimer proteins are versatile and renewableaffinity reagents." Elife.6:e24903, 2017), Affilin (Ebersbach H, et al., "Affilin-novel binding molecules based on human gamma-B-crystallin,an all beta-sheetprotein." J Mol Biol.372(1):172-185,2007), FHA domain (Durocher D, et al., "The FHAdomain." FEBS Lett. 513(1):58-66, 2002) and SH2 domains (Pawson T, et al., "SH2 domains, interaction modules and cellular wiring." Trends Cell Biol. 11(12):504-511, 2001).

[0246] Determination

[0247] Confocal microscopy.

[0248] HeLa cells were cultured at 5 x 10 4 Cells were seeded at a density of 10 cells / mL in a 35 / 10 mm glass bottom microwell dish with four compartments and cultured overnight in DMEM containing 10% FBS and 1% Ab. Cells were washed twice with DPBS and treated with 5 μM TMR-labeled proteins for 2 hours in phenol red-free DMEM containing 1% FBS and 1% Ab. Cells were washed twice with DPBS, supplemented with phenol red-free DMEM, and imaged on a Nikon A1R live cell imaging confocal microscope. Image analysis was performed using NIS ElementsAR.

[0249] Flow cytometry.

[0250] HeLa cells were cultured at 7.5 x 10 4 The density of cells / well was seeded in 24-well plates. On the day of the experiment, the cells were incubated with 5 μM TMR-labeled proteins in DMEM medium supplemented with 1% FBS and 1% Ab for 2 hours. The cells were washed with cold DPBS and harvested by trypsinization. The detached cells were washed twice with DPBS, resuspended in DPBS, and analyzed by flow cytometry (BD FACS Aria III).

[0251] Western blot.

[0252] NIH-3T3 cells were cultured at 37°C and 5% CO2 for 10 6 The density of cells / well was seeded in standard DMEM supplemented with 10% FBS and 1% Ab in 6-well plates. The cells were starved in serum-free medium for 3 hours. Cells were treated with different concentrations of MTD4-PTP1B for 4 hours and stimulated with EGF (50ng / mL) for 10 minutes. The cells were harvested, washed with PBS, and lysed on ice for 30 minutes in 100μL of Pierce RIPA buffer (Thermo) containing proteases, phosphatase inhibitors and sodium pervanadate. The lysate was centrifuged at 15,000rpm for 20 minutes. The total protein concentration of each sample was measured using a BCA protein assay kit (Thermo). Equal amounts of protein were loaded onto each lane of a 10% SDS-PAGE gel (120V, 2.5 hours). Proteins were electrophoretically transferred to a nitrocellulose membrane at 4°C (90V, 2.5 hours). The membrane was blocked with 5% BSA in TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v / v) Tween-20) for 1 hour at room temperature. Finally, the membrane was incubated with anti-pY antibody 4G10 (1:1000 dilution) at 4°C overnight. The membrane was washed three times with TBST and incubated with fluorescently labeled secondary antibodies (1:10,000 dilution) at room temperature for 2 hours. The membrane was washed again with TBST three times and the signal was acquired using a LICOR Odyssey CLx machine.

[0253] For western blotting using anti-p-Akt and anti-p-MEK antibodies. MiaPaCa-2 cells were seeded in 12-well plates at a density of 150,000 cells / well in DMEM medium supplemented with 10% FBS and 1% ABS and incubated at 37°C, 5% CO2. The next day, cells were treated with PBS or different concentrations of MTD4-RBDV or MTD4-NS1 for 4 hours, and then stimulated with EGF (50ng / mL) for 10 minutes. Cells were harvested, lysed and analyzed by western blotting using anti-p-Akt and anti-p-MEK antibodies, as described for MTD4-PTP1B.

[0254] HEK293T cells were cultured at 30 x 10 5 The density of cells / well was seeded in standard DMEM supplemented with 10% FBS and 1% Ab in 6-well plates. Cells were treated with different concentrations of MTD4-PTP1B in serum-free medium for 6 hours. The cells were harvested, washed with PBS, and lysed on ice for 30 minutes in 100 μL Prince RIPA buffer (Thermo) containing protease, phosphatase inhibitors and sodium pervanadate. The lysate was centrifuged at 15,000 rpm for 20 minutes. The total protein concentration of each sample was measured using a BCA protein assay kit (Thermo). Equal amounts of protein were loaded onto each lane of a 10% SDS-PAGE gel (120 V, 2.5 hours). Protein was electrophoretically transferred to a nitrocellulose membrane at 4 ° C (90 V, 2.5 hours). The membrane was blocked at room temperature with 5% BSA in TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v / v) Tween-20) for 1 hour. Finally, the membrane was incubated with anti-pY antibody 4G10 (1:1000 dilution) at 4°C overnight. The membrane was washed three times with TBST and incubated with fluorescently labeled secondary antibodies (1:10,000 dilution) at room temperature for 2 hours. The membrane was washed again three times with TBST and the signal was acquired using a LICOR Odyssey CLx machine.

[0255] Cell viability assay.

[0256] H358, MiaPaCa-2, H1915 or H1299 cells were seeded in white 96-well plates (5000 cells / well). The next day, cells were treated with serially diluted protein solutions or PBS. Cells were incubated at 37°C, 5% CO2 for 72 hours. After adding Cell Titer Glo and incubating for another 15 minutes, luminescence was measured on a TECAN instrument. The reported viability values ​​are relative to the viability values ​​of PBS-treated control cells.

[0257] BRET assay.

[0258] HEK293T were seeded (650,000 cells / well) in 6-well plates and incubated at 37°C, 5% CO2. The next day, cells were transfected with pEF-RLUC8-L15-KrasG12D and pEF-CRAFRBD(1-149)-L15-GFP or pEF-RLUC8-L15-KrasG12V and pEF-CRAFRBD(1-149)-L15-GFP (at a ratio of 1:2 (50ng KRAS and 100ng CRAFRBD-GFP)). Cells were incubated at 37°C for 24 hours after transfection. The next day, cells were seeded in white 96-well plates (50,000 cells / well) and incubated at 37°C for 4 hours, followed by the addition of different concentrations of MTD4-RBDV or MTD4-NS1. After 20-24 hours of incubation and addition of 10 μM coelenterazine 400a substrate, BRET signals were measured on a TECAN instrument.

[0259] Annexin-V / PI staining.

[0260] H358 cells were cultured at 10 × 10 4 The density of cells / well was inoculated into 1mL RPMI containing 10% FBS and 1% Ab in 12-well microplates and incubated overnight at 37°C and 5% CO2. The next day, the culture medium was removed and the cells were washed with DPBS, and treated with different concentrations of MTD4-RBDV or MTD4-NS1 at 37°C and 5% CO2 in 1mL DMEM culture medium containing 10% FBS for 24 hours. In order to harvest cells, the culture medium was collected in 15mL falcon tubes. The cells were washed with DPBS, and the washing solution was mixed with the culture medium in the corresponding falcon tubes. The adherent cells were treated with 250 μL of 0.25% trypsin / well at 37°C for 3 minutes and then transferred back to their respective falcon tubes. The cells were precipitated by centrifugation at 300g, 4°C for 5 minutes, and the cells were washed twice with DPBS to remove any residual trypsin. Annexin V staining was then performed according to the protocol of Invitrogen. Resuspend the cell pellet in 100 μL of 1X Annexin Binding Buffer. Next, add 5 μL of Alexa Fluor 488 Annexin V and 1 μL of propidium iodide (PI, 100 μg / ml) were added to the cell suspension and incubated for 15 min at RT. Finally, 400 μL of Annexin binding buffer was added to each tube immediately before analyzing the fluorescence emission at 530 nm and 575 nm on a BD LSR Fortessa flow cytometer.

[0261] Peptide synthesis and conjugation of HiBit peptides.

[0262] All peptides were manually synthesized on rink amide resin using standard Fmoc chemistry. A typical coupling reaction contained 5 equivalents of Fmoc-amino acid, 5 equivalents of HATU, and 10 equivalents of diisopropylethylamine (DIPEA) and was allowed to mix for 30 minutes at room temperature (RT). For CPP12, which has a single cysteine ​​at the C-terminus, the allyl group on the C-terminal Glu residue was removed by treatment with 0.3 equivalents of Pd(PPh3)4 and 10 equivalents of phenylsilane in anhydrous DCM in the dark (3×15 minutes) after the last (N-terminal) residue was added. The resin was washed twice with sodium dimethyldithiocarbamate (SDDCM, 0.5M in DMF) and the N-terminal Fmoc group was removed by treatment with 20% piperidine in DMF. The resin was washed thoroughly with DMF and DCM and incubated in 1M 1-hydroxybenzotriazole (HOBt) in DMF for 20 minutes. The peptide was cyclized at RT using 10 equivalents of PyBOP, 10 equivalents of HOBT and 20 equivalents of DIPEA in DMF for 1 hour. Cleavage and deprotection of the peptide were performed on the resin at RT for 3 hours using 92.5 / 2.5 / 2.5 / 2.5 (v / v) TFA / triisopropylsilane / 1,3-dimethoxybenzene / water. HiBit peptide (CVSGWRLFKKIS) (SEQ ID NO: 152) with N-terminal cysteine ​​was synthesized as described above. The purified HiBit peptide was activated and separated with 2,2′-bipyridyl disulfide (PyS-HiBit). For conjugation, 1 equivalent of PyS-HiBit peptide was incubated with CPP12-Cys in methanol containing 2% acetic acid, and the conjugated peptide was purified by reverse phase HPLC on a semi-preparative WatersXBridge C18 column. Similarly, 3 equivalents of PyS-HiBit were incubated with MTD for 3 h in the absence of a reducing agent, and the reaction mixture was passed through a spin desalting column to remove excess HiBit peptide. A non-reducing SDS-PAGE gel was run and conjugation was confirmed by monitoring a shift of approximately 1.3 kDa.

[0263] HiBit delivery assay.

[0264] HEK293T cells were cultured at 60 x 10 4The density of 10 cells / well was inoculated in the inoculation medium (DMEM supplemented with 10% FBS and 1% Ab) in 6-well plates. The next day, lipofectamine 2000 was used to transfect cells with 0.5 μg LgBit plasmid DNA for 24 hours. Cells were re-inoculated in the inoculation medium in 96-well plates at a density of 10,000 cells / well overnight. Cells were treated with different concentrations of conjugated or unconjugated HiBit peptides in the culture medium supplemented with 1% FBS and 1% Ab for 4 hours. The cells were washed twice with DPBS, and 100 μL OPTIMEM and 25 μL NanoLuc reagents were added to each well afterwards. Tecan Infinite M1000 Pro microplate reader was used to measure luminescence immediately. These values ​​were normalized relative to the values ​​of untreated cells, and GraphPad Prism software was used to plot the mean ± SD for peptide concentration.

[0265] GFP complementation assay.

[0266] HEK293T cells were cultured at 60 x 10 4 The cells were seeded at a density of 10 cells / well in a 6-well plate in medium supplemented with 10% FBS and 1% Ab. The next day, the cells were transfected with 1 μg of GFP1-10 plasmid DNA using lipofectamine 2000 for 24 hours. The cells were plated at 5 x 10 4 Cells were seeded at a density of 10 cells / mL in a 35 / 10 mm glass-bottom microwell dish with four compartments and cultured overnight in DMEM containing 10% FBS and 1% Ab. Cells were washed twice with DPBS and treated with DPBS (no treatment control) or 10 μM GFP11, CPP12-GFP11 or MTD4-GFP11 for 6 hours in a medium supplemented with 1% FBS and 1% Ab. Cells were washed twice with DPBS, supplemented with phenol red-free medium, and imaged on a Nikon A1R confocal microscope. Image analysis was performed using NIS Elements AR.

[0267] Biodistribution studies.

[0268] All animal experiments were performed in accordance with institutional animal care guidelines and committee-approved protocols. Endotoxins were removed from concentrated MENC protein using Pierce high-capacity endotoxin removal resin. Endotoxin-free MENC protein (8 mg / kg) was intravenously injected into 4 floxed mice (transgenic mice with loxP modification) through the tail vein. Two mice were killed after 3 hours, and the other two mice were killed after 48 hours. Different organs were harvested, fixed in 4% paraformaldehyde for 24 hours, and then transferred to 70% ethanol solution. The fixed samples were sent to iHisto Inc. for embedding, sectioning and slide preparation. The slides were washed twice with 100% Xxylene, 100% ethanol, 95% ethanol and ddH2O for imaging. The slides were stained with pure black dye (Biotium), sealed and sealed with toluene. The slides were imaged on a Nikon A1R confocal microscope and analyzed using NIS Elements AR software.

[0269] Serum stability test.

[0270] FN3 or MTD4 (10 μM) was incubated with 25% clarified human serum in a total volume of 100 μL. The mixture was incubated at 37°C and 10 μL aliquots were taken at different time points. The aliquots were immediately mixed with 10 μL of 2x SDS loading buffer, boiled for 5 minutes, and stored at -20°C. After incubation for up to 24 hours, all aliquots were analyzed on 15% SDS-PAGE gels and the gels were stained with Coomassie Blue. The gel was scanned in the 700nm channel on an Odyssey CLx imager (LI-COR).

[0271] sequence

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

Claims

1. A peptide comprising: a membrane translocation domain having one or more cell penetrating peptide motifs, wherein at least one cell penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least three arginine and / or lysine residues; or wherein at least one cell penetrating peptide motif has a length of 3 to 10 amino acid residues and has at least two arginine and / or lysine residues, and at least one other cell penetrating peptide motif has a length of 2 to 8 amino acid residues and has at least two hydrophobic residues.

2. The peptide of claim 1, wherein the membrane translocation domain is a mammalian membrane translocation domain.

3. The peptide according to any one of the preceding claims, wherein the membrane translocation domain is human type III fibronectin.

4. The peptide according to any one of the preceding claims, wherein the human type III fibronectin has 90% sequence similarity to SEQ.ID.NO.

118.

5. The peptide according to any one of the preceding claims, wherein the cell penetrating motif has 3 to 10 consecutive arginine residues.

6. A peptide according to any one of the preceding claims, wherein the membrane translocation domain is human type III fibronectin having BC, DE, CD and FG loops, and one or more of the BC, DE, CD or FG loops have a cell penetrating peptide motif.

7. The peptide according to any one of the preceding claims, wherein the membrane translocation domain is human type III fibronectin having BC, DE, CD and FG loops, and two of the BC, DE, CD or FG loops have a cell penetrating peptide motif.

8. The peptide according to any one of the preceding claims, wherein the membrane translocation domain is human type III fibronectin having BC, DE, CD and FG loops, and the BC loop and any one of the DE, CD or FG loops have a cell penetrating peptide motif.

9. The peptide according to any one of the preceding claims, wherein the membrane translocation domain is human type III fibronectin having BC, DE, CD and FG loops, and the BC and FG loops have a cell penetrating peptide motif.

10. The peptide according to claim 9, wherein the cell penetrating peptide motif in the BC loop has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the cell penetrating peptide motif in the FG loop has 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

11. The peptide according to any one of the preceding claims, wherein the cell penetrating peptide motif in the FG loop has 2 to 8 amino acid residues and has at least two hydrophobic amino acid residues, and the cell penetrating peptide motif in the BC loop has 3 to 10 amino acid residues and has at least two adjacent arginine and / or lysine residues.

12. The peptide of any one of the preceding claims, wherein a second cell penetrating peptide motif is present and is WW, FF, WF, FW, WWW, FFF, WFW, FWF, WWF, WFF, FWW, FFW, WYW, WWH, YWW or WYH.

13. The peptide according to any one of the preceding claims, wherein the cell penetrating peptide motif is RRRWWW (SEQ.ID.NO.: 104) or WWWRRR (SEQ.ID.NO.: 105).

14. A peptide according to any one of the preceding claims, comprising TGRRRRWWWSKPI (SEQ.ID.NO.: 111); APWWWRRRRYY (SEQ.ID.NO.: 112); GGRRRRWWWVQE (SEQ.ID.NO.: 113); APAWYWRYY (SEQ.ID.NO.: 114); TGRRRRSKPI (SEQ.ID.NO.: 115); APARRRRYY (SEQ.ID.NO.: 116); or TGWYWRSKPI (SEQ.ID.NO.: 117).

15. A peptide according to any one of the preceding claims, comprising SEQ.ID.NO.: 119, 120, 121, 122, 123, 124 or 125.

16. The peptide of any one of the preceding claims, further comprising a cargo moiety linked to the membrane translocation domain.

17. The peptide of claim 16, wherein the cargo moiety is linked to the membrane translocation domain at the N-terminus or C-terminus of the membrane translocation domain or at a side chain within a membrane translation domain.

18. The peptide of any one of claims 16 or 17, wherein the cargo moiety is selected from the group consisting of a detectable moiety, a targeting moiety, a therapeutic moiety, or any combination thereof.

19. A peptide according to any one of the preceding claims, comprising SEQ.ID.NO.: 126, 127 or 128.

20. A composition for delivering a cargo moiety into a cell, comprising SEQ.ID.NO.: 122 covalently bound to a cargo, wherein the cargo moiety is selected from the group consisting of a detectable moiety, a targeting moiety, a therapeutic moiety, or any combination thereof.

21. A method of delivering a cargo moiety into a cell, comprising contacting the cell with a peptide according to any one of the preceding claims.

22. A method of delivering an agricultural product into a plant cell comprising contacting the cell with a peptide according to any one of the preceding claims.

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