Cell penetrating peptides and methods of making and using the same

Abstract

Disclosed herein are compounds having activity as cell penetrating peptides. In some instances, the compound may comprise a cell penetrating peptide portion and a cargo portion. The cargo portion may comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof. In some instances, the cell penetrating peptide portion is cyclic. In some instances, the cell penetrating peptide portion and the cargo portion are cyclic together. In some instances, the cell penetrating peptide portion is cyclic, and the cargo portion is attached to a cyclic cell penetrating peptide portion structure. In some instances, the cargo portion is cyclic, and the cell penetrating peptide portion is cyclic, and they form a fused bicyclic system together.

Description

[0001] The present application is a divisional application of the application with application number 201580034608.9, application date May 21, 2015, and invention name “Cell Penetrating Peptide and Method for Preparation and Use thereof”. background

[0002] The plasma membrane is a major challenge in the development of drugs, especially biologics such as peptides, proteins, and nucleic acids. A potential strategy to disrupt the membrane barrier and deliver biologics into cells is to connect biologics to "cell penetrating peptides (CPPs)". Despite three decades of research, the fundamental basis of CPP activity remains unclear. CPPs that enter cells via endocytosis must be expelled from endocytic vesicles to reach the cytoplasm. Unfortunately, the endosomal membrane has been shown to be an important barrier to cytoplasmic delivery by these CPPs; a negligible portion of the peptides usually escape into the cell interior (El-Sayed, A et al., AAPS J., 2009, 11, 13-22; Varkouhi, AK et al., J. Controlled Release, 2011, 151, 220-228; Appelbaum, JS et al., Chem. Biol., 2012, 19, 819-830). Therefore, novel cell penetrating peptides and compositions comprising such peptides that can be used to deliver agents to various cell types are needed. The compositions and methods disclosed herein address these and other needs.

[0003] Overview

[0004] Disclosed herein are compounds having activity as cell penetrating peptides. In some examples, the compound may comprise a cell penetrating peptide portion and a cargo moiety. The cargo moiety may comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof.

[0005] In some instances, the cell penetrating peptide portion is cyclic. In some instances, the cell penetrating peptide portion and the cargo portion are cyclic together; this is referred to herein as an "endocyclic" configuration. In some instances, the cell penetrating peptide portion is cyclic, and the cargo portion is attached to the cyclic cell penetrating peptide portion structure; this is referred to herein as an "exocyclic" configuration. In some instances, the cargo portion is cyclic, and the cell penetrating peptide portion is cyclic, and together they form a fused bicyclic system; this is referred to herein as a "bicyclic" configuration.

[0006] In some examples, the compound can have Formula I:

[0007]

[0008] Among them, AA 1AA 2 AA 3 AA 4 AA 5 AA 6 AA 7 AA 8 and AA 9 (i.e., AA 1 -AA 9 ) are each independently an amino acid; and m, n and p are independently selected from 0 and 1. In other examples of Formula I, there may be more than 9 amino acids, such that when m and p are 1, n is 2 or greater. These larger peptides are disclosed in various formulas herein, such as IA, II, IIa, IIb and IIc. In some examples, three or more amino acids are arginine, and one or more are phenylalanine. In other examples, one or more amino acids are naphthylalanine or tryptophan.

[0009] In some examples, the cell penetrating peptide portion is cyclic, and the compound can be a compound of Formula Ia:

[0010]

[0011] Among them, AA 1 -AA 9 , m, n and p are as defined in Formula I, and wherein the curved line represents a covalent bond.

[0012] In some examples, the compound further comprises a cargo moiety, and the compound can have Formula II:

[0013]

[0014] wherein the cargo moiety may comprise a detectable moiety, a therapeutic moiety, a targeting moiety, or a combination thereof, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0015] In some examples, the cell penetrating peptide moiety and the cargo moiety together are cyclic, and the compound has Formula IIa:

[0016]

[0017] wherein the cargo moiety is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0018] In some examples, the cell penetrating peptide moiety is cyclic, and the cargo moiety is attached to the cyclic cell penetrating peptide moiety structure, and the compound has Formula IIb:

[0019]

[0020] Wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0021] In some examples, the cargo moiety is cyclic and the cell penetrating peptide moiety is cyclic, the cargo moiety and the cell penetrating peptide moiety together form a fused bicyclic ring system, and the compound has Formula IIc:

[0022]

[0023] Wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0024] The amino acid can be coupled via a peptide bond.The amino acid can be coupled to the cargo moiety at the amino group, the carboxylate group or the side chain.

[0025] In some instances, at least one amino acid comprises naphthylalanine or an analog or derivative thereof. In some instances, at least three amino acids independently comprise arginine or an analog or derivative thereof. In some instances, at least one amino acid comprises phenylalanine or an analog or derivative thereof. In some instances, at least one amino acid comprises glutamine or an analog or derivative thereof.

[0026] In some examples, the cell penetrating peptide portion can be any one of SEQ ID NO: 1 to SEQ ID NO: 90. In some examples, the cell penetrating peptide portion can be a variant of any one of SEQ ID NO: 1 to SEQ ID NO: 90.

[0027] The cargo moiety can include any cargo of interest, such as a linker moiety, a detectable moiety, a therapeutic moiety, a targeting moiety, etc., or any combination thereof.

[0028] The cargo moiety can be linked to the cell penetrating peptide moiety at the amino group, carboxylate group, or side chain of any amino acid of the cell penetrating peptide moiety (e.g., at the amino group, carboxylate group, or side chain or AA 1 -AA 9 any one of ).

[0029] In some instances, the therapeutic moiety comprises a targeting moiety. The targeting moiety may comprise, for example, an amino acid sequence that can target one or more enzyme domains. In some instances, the targeting moiety may comprise an inhibitor for a protein that may play a role in diseases such as cancer, cystic fibrosis, diabetes, obesity, or a combination thereof. In some instances, the therapeutic moiety may comprise a targeting moiety that can be used as an inhibitor for Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, etc., or a combination thereof.

[0030] Also disclosed herein are compositions comprising the compounds described herein.Also disclosed herein are pharmaceutically acceptable salts and prodrugs of the disclosed compounds.

[0031] 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.

[0032] Also provided herein are methods for treating, preventing or ameliorating cancer in a subject. The method comprises administering to the subject an effective amount of one or more compounds or compositions described herein or a pharmaceutically acceptable salt thereof. The methods for treating or preventing cancer described herein may also comprise treatment with one or more additional agents (e.g., anticancer agents or ionizing radiation).

[0033] 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, provided herein is a method of tumor radiotherapy. 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.

[0034] 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 can be used as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or a combination thereof.

[0035] The disclosed subject matter also relates to methods for treating subjects with metabolic disorders or diseases. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject suffering from a metabolic disorder and in need of such treatment. In some examples, the metabolic disorder may include type II diabetes. In some examples of methods of treating, preventing or ameliorating 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 that can be used as an inhibitor against PTP1B.

[0036] The disclosed subject matter also relates to methods for treating subjects with immune disorders or diseases. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject suffering from an immune disorder and in need of such treatment. In some examples of therapeutic methods for treating, preventing or ameliorating an immune disorder in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can be used as an inhibitor against Pin1.

[0037] 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 such treatment. In some examples of methods of treating cystic fibrosis in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can act as an inhibitor against CAL PDZ.

[0038] The present invention also includes the following items:

[0039] 1. A cell penetrating peptide compound of formula I:

[0040]

[0041] Among them, AA 1 AA 2 AA 3 AA 4 AA 5 AA 6 AA 7 AA 8 and AA 9 each is independently an amino acid; and m, n and p are independently selected from 0 and 1, wherein three or more of the amino acids are arginine.

[0042] 2. The cell penetrating peptide compound according to item 1, wherein m, n and p are 0 and the compound has formula I-1:

[0043] AA 1 -AA2 -AA 3 -AA 4 -AA 5 -AA 6

[0044] I-1

[0045] Among them, AA 1 -AA 6 As defined in Formula I.

[0046] 3. The cell penetrating peptide compound according to item 1, wherein m is 1, and n and p are 0, and the compound has formula I-2:

[0047] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7

[0048] I-2

[0049] Among them, AA 1 -AA 7 As defined in Formula I.

[0050] 4. The cell penetrating peptide compound according to item 1, wherein m and n are 1, p is 0, and the compound has formula I-3:

[0051] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8

[0052] I-3

[0053] Among them, AA 1 -AA 8 As defined in Formula I.

[0054] 5. The cell penetrating peptide compound according to item 1, wherein m, n and p are 1, and the compound has formula I-4:

[0055] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7-AA 8 -AA 9

[0056] I-4

[0057] Among them, AA 1 -AA 9 As defined in Formula I.

[0058] 6. The cell penetrating peptide according to any one of items 1 to 5, wherein the cell penetrating peptide compound is cyclic.

[0059] 7. The cell-penetrating peptide compound according to item 1, wherein the cell-penetrating peptide compound is cyclic and has formula Ia:

[0060]

[0061] Among them, AA 1 -AA 9 , m, n and p are as defined in Formula I, and wherein the curved line represents a covalent bond.

[0062] 8. The cell penetrating peptide compound according to item 7, wherein m, n and p are 0 and the compound has formula Ia-1:

[0063]

[0064] Among them, AA 1 -AA 6 As defined in Formula I.

[0065] 9. The cell penetrating peptide compound according to item 7, wherein m is 1, n and p are 0, and the compound has formula Ia-2:

[0066]

[0067] Among them, AA 1 -AA 7 As defined in Formula I.

[0068] 10. The cell penetrating peptide compound according to item 7, wherein m and n are 1, p is 0, and the compound has formula Ia-3:

[0069]

[0070] Among them, AA 1 -AA 8 As defined in Formula I.

[0071] 11. The cell penetrating peptide compound according to item 7, wherein m, n and p are 1, and the compound has formula Ia-4:

[0072]

[0073] Among them, AA 1 -AA 9 As defined in Formula I.

[0074] 12. The cell penetrating peptide compound according to any one of items 1-11, wherein the cell penetrating peptide compound further comprises a cargo portion, wherein the cargo portion may comprise a detectable portion, a therapeutic portion, a targeting portion, or a combination thereof.

[0075] 13. The cell penetrating peptide compound according to item 1, wherein the cell penetrating peptide compound has formula II:

[0076]

[0077] wherein the cargo moiety may comprise a detectable moiety, a therapeutic moiety, a targeting moiety, or a combination thereof, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0078] 14. The cell penetrating peptide compound according to item 13, wherein m, n and p are 0 and the compound has formula II-1:

[0079] AA 1- AA 2 -AA 3 -AA 4 -AA 5 -AA 5 -goods

[0080] II-1

[0081] Among them, AA 1 -AA 6 As defined in Formula I, and the cargo is as defined in Formula II.

[0082] 15. The cell penetrating peptide compound according to item 13, wherein m is 1, and n and p are 0, and the compound has formula II-2:

[0083] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -goods

[0084] II-2

[0085] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0086] 16. The cell penetrating peptide compound according to item 13, wherein m and n are 1, p is 0, and the compound has formula II-3:

[0087] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -goods

[0088] II-3

[0089] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0090] 17. The cell penetrating peptide compound according to item 13, wherein m, n and p are 1 and the compound has formula II-4:

[0091] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -AA 9 -goods

[0092] II-4

[0093] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0094] 18. The cell penetrating peptide compound according to any one of items 12 to 16, wherein the cell penetrating peptide portion and the cargo portion together are cyclic.

[0095] 19. The cell penetrating peptide compound according to item 13, wherein the cell penetrating peptide compound has formula IIa:

[0096]

[0097] wherein the cargo moiety is as defined in Formula II and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0098] 20. The cell penetrating peptide compound according to item 19, wherein m, n and p are 0 and the compound has formula IIa-1:

[0099]

[0100] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0101] 21. The cell penetrating peptide compound according to item 19, wherein m is 1, and n and p are 0, and the compound has formula IIa-2:

[0102]

[0103] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0104] 22. The cell penetrating peptide compound according to item 19, wherein m and n are 1, p is 0, and the compound has formula IIa-3:

[0105]

[0106] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0107] 23. The cell penetrating peptide compound according to item 19, wherein m, n and p are 1, and the compound has formula IIa-4:

[0108]

[0109] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0110] 24. The cell penetrating peptide compound according to any one of items 12-16, wherein the cell penetrating peptide portion is cyclic and the cargo portion is attached to the cyclic cell penetrating peptide portion.

[0111] 25. The cell penetrating peptide compound according to item 13, which has formula IIb:

[0112]

[0113] wherein the cargo moiety is as defined in Formula II and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0114] 26. The cell penetrating peptide compound according to item 25, wherein m, n and p are 0 and the compound has formula IIb-1:

[0115]

[0116] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0117] 27. The cell penetrating peptide compound according to item 25, wherein m is 1, and n and p are 0, and the compound has formula IIb-2:

[0118]

[0119] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0120] 28. The cell penetrating peptide compound according to item 25, wherein m and n are 1, p is 0, and the compound has formula IIb-3:

[0121]

[0122] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0123] 29. The cell penetrating peptide compound according to item 25, wherein m, n and p are 1, and the compound has formula IIb-4:

[0124]

[0125] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0126] 30. The cell penetrating peptide compound according to any one of items 12-16, wherein the cargo portion is cyclic and the cell penetrating peptide portion is cyclic, and together they form a fused bicyclic system.

[0127] 31. A cell penetrating peptide compound according to item 13, wherein the compound has formula IIc:

[0128]

[0129] wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0130] 32. The cell penetrating peptide compound according to item 31, wherein m, n and p are 0 and the compound has formula IIc-1:

[0131]

[0132] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0133] 33. The cell penetrating peptide compound according to item 31, wherein m is 1, and n and p are 0, and the compound has formula IIc-2:

[0134]

[0135] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0136] 34. The cell penetrating peptide compound according to item 31, wherein m and n are 1, p is 0, and the compound has formula IIc-3:

[0137]

[0138] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0139] 35. The cell penetrating peptide compound according to item 31, wherein m, n and p are 1, and the compound has the formula IIc-4:

[0140]

[0141] Among them, AA 1 -AA 6 is as defined in formula I and the cargo is as defined in formula II.

[0142] 36. The cell penetrating peptide compound according to any one of items 1 to 35, wherein at least one amino acid is naphthylalanine, tryptophan or phenylalanine or a derivative or analogue thereof.

[0143] 37. The cell penetrating peptide compound according to any one of items 1 to 36, wherein at least three of the amino acids are arginine.

[0144] 38. The cell penetrating peptide compound according to any one of items 1 to 37, wherein at least one of the amino acids is phenylalanine.

[0145] 39. A cell penetrating peptide compound according to any one of items 12-38, wherein the cargo portion comprises a therapeutic portion.

[0146] 40. The cell penetrating peptide compound according to item 39, wherein the therapeutic moiety may comprise a targeting moiety that can be used as an inhibitor against Ras, PTP1B, Pin1, Grb2 SH2, CAL PDZ, etc. or a combination thereof.

[0147] 41. A composition comprising a compound according to any one of items 1-40.

[0148] 42. A pharmaceutical composition comprising a compound according to any one of items 1-41.

[0149] 43. A method for treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a compound or composition according to any one of items 1-42.

[0150] 44. A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a compound or composition according to any one of items 1-43.

[0151] 45. A method according to any one of item 44, further comprising administering a second compound or composition, wherein the second compound or composition comprises an anti-cancer agent.

[0152] 46. ​​A method according to any one of items 44-45, further comprising administering an effective amount of ionizing radiation to the subject.

[0153] 47. A method according to any one of items 44-46, wherein the compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety that can be used as an inhibitor against Ras, PTP1B, Pin1, Grb2 SH2 or a combination thereof.

[0154] 48. A method of killing tumor cells in a subject, comprising: contacting the tumor cells with an effective amount of a compound or composition according to any one of items 1-42.

[0155] 49. The method of claim 48, further comprising contacting the tumor cells with a second compound or composition, wherein the second compound or composition comprises an anti-cancer agent.

[0156] 50. A method according to any one of items 48-49, further comprising irradiating the tumor cells with an effective amount of ionizing radiation.

[0157] 51. A method according to any one of items 49-50, wherein the compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety that can be used as an inhibitor against Ras, PTP1B, Pin1, Grb2 SH2 or a combination thereof.

[0158] 52. A method for treating a subject suffering from a metabolic disorder or condition, comprising administering an effective amount of a compound or composition according to any one of items 1-42.

[0159] 53. A method according to claim 52, wherein the metabolic disorder is type 2 diabetes.

[0160] 54. The method according to any one of items 52-53, wherein the compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety that can act as an inhibitor against PTP1B.

[0161] 55. A method for treating a subject having an immune disorder or condition, comprising administering an effective amount of a compound or composition according to any one of items 1-42.

[0162] 56. The method of claim 55, wherein the compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety that can act as an inhibitor against Pin1.

[0163] 57. A method for treating a subject having cystic fibrosis, comprising administering an effective amount of a compound or composition according to any one of items 1-42.

[0164] 58. A method according to item 57, wherein the compound or composition administered to the subject may comprise a therapeutic moiety, which may comprise a targeting moiety that can act as an inhibitor against CAL PDZ.

[0165] 59. A method according to claim 57, wherein a molecule that corrects CFTR function is also administered together with the compound or composition.

[0166] 60. A method of delivering a compound to a cardiomyocyte, comprising: contacting the cardiomyocyte with an effective amount of the compound or composition according to any one of items 1-42.

[0167] 61. A compound as shown in any one of Tables 1, 6 or 18.

[0168] 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 will be apparent from the description and drawings, and from the claims.

[0169] Description of the drawings

[0170] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0171] Figure 1 shows the structures of cargo attachment during intra-ring (A), extra-ring (B), and bi-ring delivery (C) of cargo (shown in light grey) by cFΦR4.

[0172] Figure 2 Shown are the structures of some of the peptides used in this study.

[0173] Figure 3 Shown is a scheme illustrating the synthesis of cFΦR4-SS-GFP.

[0174] Figure 4 A scheme showing the synthesis of cFΦR4-PTP1B is shown.

[0175] FIG5 shows the binding of FITC-labeled cFΦR4, R9 and Tat to (A) SUV and (B) heparan sulfate.

[0176] Figure 6 shows peptides labeled with rhodamine B and the fluid phase uptake marker dextran FITC Representative live cell confocal images of HEK293 cells treated for 2 hours. (A) FITC (B) 5 μM cFΦR4-R5 and dextran were used to treat the cells in the same Z-section. FITC Processed cells in the same Z-section.

[0177] Figure 7 cFΦR4 showed that HeLa cells were sensitive to dextran Alexa488 HeLa cells were treated with no supplement, 1 μM cFΦR4, 100 μM dextran,Alexa488 or 1 μM cFΦR4 and 100 μM dextran Alexa488 of clarified DMEM. MFI, mean fluorescence intensity.

[0178] Figure 8 The effect of pH on CAP fluorescence is shown. cFΦR4-PCP was dephosphorylated by alkaline phosphatase and purified by HPLC, and its fluorescence at the indicated pH was measured.

[0179] Figure 9 shows the internalization of pCAP-containing peptides into cultured cells: I, unlabeled PCP; II, cFΦR4-PCP; III, cFΦR4-PCP and Na3VO4; IV, R9-PCP; V, Tat-PCP; and VI, Antp-PCP. (A) Representative live cell confocal images of HEK293 cells treated with 5 μM peptide. Upper panel, nuclear staining using DRAQ5; lower panel, CAP fluorescence in the same Z-section. (B) Flow cytometry of HeLa cells treated with 0 or 10 μM peptide. (C) CAP fluorescence after subtracting background fluorescence (untreated cells) from (B). MFI, mean fluorescence intensity.

[0180] Figure 10 shows the peptides labeled with rhodamine B (all at 5 μM) and the fluid phase endocytosis marker dextran FITC Representative live cell confocal microscopy images of HEK293 cells treated with 0.5 mg / mL of dapoxetine for 2 hours. Each image shows the red fluorescence of rhodamine B and dextran from the same Z-section. FITC Green fluorescence and its merged image. In each case, a magnified image of a typical cell is shown to illustrate the intracellular distribution of the internalized peptide. (A) Using bicyclic (F ΦR4-A5) Rho Treated cells; (B) Single ring (FΦR4-A5) Rho ; (C) Bicyclic (F ΦR4-A7) Rho ; (D) Single ring (FΦR4-A7) Rho ; (E) Bicyclic (FΦR4-RARAR) Rho ; and (F) bicyclic (FΦR4-DADAD) Rho .

[0181] Figure 11 shows (A) the structure of CPP-SS-GFP conjugates. (B) Live cell confocal images of mammalian cells treated with 1 μM GFP (I), Tat-SS-GFP (II) or cFΦR4-SS-GFP (III) and nuclear stain DRAQ5 for 2 hours. All images were recorded at the same Z section.

[0182] Figure 12 shows (A) Western blot analysis of global pY protein levels in NIH3T3 cells after treatment with 0-500 nM PTP1B or cFΦR4-PTP1B (IB: anti-pY antibody 4G10). (B) The same samples as in (A) were analyzed by SDS-PAGE and Coomassie blue staining. M, molecular weight marker.

[0183] Figure 13 shows (A) Comparison of serum stability of cFΦR4, Tat, R9 and Antp. (B) Cytotoxicity of cFΦR4. The indicated cell lines were treated with DMSO (control), 5 μM or 50 μM cFΦ R4 for 24 hours, and the percentage of viable cells was determined by MTT assay.

[0184] FIG. 14 shows MTT assays of various mammalian cells after treatment with cFΦR4 (5 or 50 μM) for (A) 48 hours or (B) 72 hours.

[0185] Fig.15 Shown is a diagram showing points along the endocytic pathway where cFΦR4, R9 and Tat escape into the cytoplasm and where specific inhibitors are proposed to act.

[0186] Fig.16 Shown is a scheme illustrating a reversible cyclization strategy for delivering linear peptidyl cargoes into mammalian cells. GSH, glutathione.

[0187] Figure 17 shows (A) Synthesis of disulfide bond cyclized peptides. (B) Synthesis of thioether bond cyclized peptides. Reagents and conditions: (a) Standard Fmoc / HATU chemistry; (b) Piperidine / DMF; (c) 3,3'-dithiodipropionic acid / DIC; (d) β-mercaptoethanol / DMF; (e) Modification reagent K; (f) Grinding; (g) DMSO / DPBS (pH 7.4). (h) 4-bromobutyric acid / DIC; (i) 1% TFA / DCM; (j) 1% DIPEA / DMF; PG, protecting group. Trt, trityl; Mmt, methoxytrityl. (C) Structure of FITC-labeled peptides 1 and 2. (D) Structure of pCAP (phosphocoumarylaminopropionic acid) containing peptides 1-PCP and 2-PCP. (E) Structure of Amc (7-amino-4-methylcoumarin) containing caspase fluorescent substrate 3-7. (F) Structure of FITC-labeled CAL-PDZ domain ligands 9-11.

[0188] FIG18 shows (A) the expression of peptides 1 (I) or 2 (II) labeled with 5 μM FITC, the endocytosis marker dextran Rho (0.5 mg mL -1(A) Live cell confocal microscopy images of HeLa cells treated with 5 μM FITC-labeled peptides 1, 2 or FITC alone. Images in different fluorescence channels were recorded at the same Z section. (B) Flow cytometry of HeLa cells treated with 5 μM FITC-labeled peptides 1, 2 or FITC alone.

[0189] Figure 19 shows (A) FACS analysis of HeLa cells treated with 0 or 5 μM peptides 1-PCP, 2-PCP for 2 hours. (B) CAP fluorescence after subtracting background fluorescence (untreated cells) from (A). MFI, mean fluorescence intensity.

[0190] Fig. 20 Comparison of the proteolytic stability of peptides 1 and 2 is shown.

[0191] Fig.21 Shown is the time-dependent release of the fluorescent coumarin product from Jurkat cells treated with peptide 3-7 (5 μM) in the absence and presence of 100 μM caspase inhibitor Z-VAD(OMe)-FMK(FMK).

[0192] Figure 22 shows (A) the structure of the CAL-PDZ inhibitor 8. (B) Binding of peptide 8 to the CAL-PDZ domain in the presence or absence of a reducing agent. (C) Live cell microscopy images of HeLa cells treated with peptide 8 (5 μM) and DRAQ5 at the same Z-section. I, green fluorescence of internalized peptide 8; II, overlap of green peptide fluorescence and blue nuclear staining. (D) Immunofluorescence staining showing the distribution of CFTR in the presence or absence of Corr-4a (10 μM) and unlabeled peptide 8 (50 μM). (E) SPQ assay showing an increase in the slope of CFTR-specific stimulation-induced fluorescence in the presence or absence of VX809 (20 μM) and peptide 8 (50 μM). P values ​​were calculated by two-tailed t-test.

[0193] Fig.23 Schematic diagram showing the evolution of cell-permeable PTP1B inhibitors.

[0194] Fig.24 A schematic diagram of the design and synthesis of the cyclic peptide library is shown. Reagents and conditions: (a) standard Fmoc / HBTU chemistry; (b) immersion in water; (c) 0.1 eq Fmoc-Glu(δ-NHS)-OAll, 0.4 eq Boc-Met-OH in Et2O / CH2Cl2; (d) piperidine; (e) split into two parts; (f) synthesized by splitting and combining via Fmoc / HATU chemistry; (g) Pd(PPh3)4; (h) PyBOP, HOBt; and (i) reagent KX2 ,10% F2Pmp and 90% Tyr; X 1 and X 3 -X 5 , random position; Φ, L-2-naphthylalanine; CPP, cell penetrating motif F ΦR4 or R4ΦF.

[0195] Fig.25 Competitive inhibition of PTP1B by the monocyclic peptide inhibitor 2 is shown. (A) Lineweaver-Burk plot of PTP1B-catalyzed hydrolysis of pNPP (0-24 mM) in the presence of different concentrations of inhibitor 2 (0, 22.5, 45 and 90 nM). (B) Quadratic plot of the ratio of the Michaelis constant (K / KO) as a function of [I].

[0196] Fig.26 Inhibitor 2 (upper panel) or 4 (lower panel) labeled with 5 μM FITC and the endocytosis marker dextran are shown. Rho (a) Live cell confocal microscopy images of A549 lung cancer cells after 2 h of treatment with 4 (1.0 mg / mL) (same Z-section). (b) Lineweaver-Burk plot showing competitive inhibition of PTP1B by 0, 28, 56 and 112 nM inhibitor 4. (c) Sensitivity of various PTPs to inhibition by inhibitor 4 (all activities relative to the absence of inhibitor).

[0197] Fig. 27 Shown is the solid phase synthesis of inhibitor 4. Reagents and conditions: a) standard Fmoc chemistry; b) trimesic acid, HBTU; c) Pd(PPh3)4, N-methylaniline; d) PyBOP; e) TFA.

[0198] Fig.28 Comparison of serum stability of the monocyclic PTP1B inhibitor 2 and the bicyclic inhibitor 4 is shown.

[0199] Figure 29 is a graph of global pY protein levels in A549 cells after 2-hour treatment with 0-5 μM inhibitor 4. (b) SDS-PAGE analysis (Coomassie blue staining) of the same samples from (a) showing uniform sample loading in all lanes. (c) Effect of inhibitor 4 on Tyr 1162 and Tyr 1163 HepG2 cells were treated with the indicated concentrations of inhibitor 4 for 2 h, then stimulated with insulin (100 nM) for 5 min, and then incubated with anti-IRpY 1162 / pY 1163Antibodies were used for SDS-PAGE and immunoblotting. (d) Quantification of IR pY levels from (c) (data shown are mean ± SD from five independent experiments).

[0200] Fig.30 The impermeable Pin1 inhibitor is shown to be converted into a cell-permeable bicyclic inhibitor.

[0201] Fig.31 FA analysis showing the binding of Pin1 inhibitors 5-9 to Pin1.

[0202] Figure 32 shows competition of inhibitors 5 and 7 for binding to Pin 1. Each reaction contained 0.1 μM FITC-labeled inhibitor 5, 1 μM Pin 1, and 0-5 μM unlabeled inhibitor 5 (a) or inhibitor 7 (b), and FA values ​​were measured and plotted against competitor concentration.

[0203] Figure 33 shows the cellular uptake of Pin1 inhibitors. (a, b) Pin1 inhibitors 5 (a) or 7 (b) labeled with 5 μM FITC and 1 mg / mL endocytosis marker dextran Rho Live cell confocal microscopy images of HEK293 cells treated for 2 h. All images were recorded at the same Z section. (c) FACS analysis of HeLa cells treated for 2 h with DMSO or 5 μM FITC-labeled Pin1 inhibitors 5, 7, 8, or 9. MFI, mean fluorescence intensity. Procedure: In a six-well plate (2 × 10 5 Hela cells were cultured in 4% paraformaldehyde (40% paraformaldehyde) for 24 h. On the day of the experiment, cells were incubated with 5 μM FITC-labeled bicyclic peptides or control monocyclic peptides in phenol red-free DMEM supplemented with 1% FBS. After 2 h, the peptide solution was removed, the cells were washed with DPBS, treated with 0.25% trypsin for 5 min, and washed again with DPBS. Finally, the cells were resuspended in flow cytometry buffer and analyzed by flow cytometry (BD FACS Aria) with excitation at 535 nm.

[0204] Fig.34 The effects of Pin1 inhibitors 5, 7, 8, and 9 on cancer cell proliferation were shown. HeLa cells (100 μL per well, 5 × 10 4Cells / mL) were seeded in 96-well culture plates and grown overnight in DMEM supplemented with 10% FBS. Different concentrations of Pin1 inhibitor (0-5 μM) were added to the wells, and the cells were incubated at 37°C with 5% CO2 for 72 hours. Afterwards, 10 μL of MTT stock solution (5 mg / mL) was added to each well. The plate was incubated at 37°C for 4 hours, and 100 μL of SDS-HCl solubilization solution was added to each well and then mixed thoroughly. The plate was incubated at 37°C overnight, and the absorbance of the formazan product was measured at 570 nm on a Molecular Devices Spectramax M5 plate reader. Each experiment was performed in triplicate, and cells not treated with peptide were used as controls.

[0205] Figure 35 shows live cell confocal images of mouse ventricular cardiomyocytes treated with 5 μM c(FΦRRRRQ)-K(FITC) (a) and c(fΦRrRrQ)-K(FITC) (b) for 3 hours. (c) Calmodulin labeled with a cyclic cell penetrating peptide via a disulfide bond (T5C). (d) Live cell confocal images of mouse ventricular cardiomyocytes treated with 6 μM cFΦR4-conjugated Cy3-labeled calmodulin for 3 hours.

[0206] Fig.36 Shown is the progress of a bicyclic peptide inhibitor against Pin 1. The parts of the structure derived from the library screen are shown in grey, while changes made during optimization are shown in light grey.

[0207] Figure 37 shows characterization of peptide 37. (a) Binding of FITC-labeled peptide 37 to Pin1 analyzed by fluorescence anisotropy (FA). (b) Competition of peptide 37 and FITC-labeled peptide 1 (100 nM) for binding to Pin1 (400 nM) monitored by FA. (c) Effect of peptide 37 on the cis-trans isomerase activity of Pin1, Pin4, FKBP12 and cyclophilin A using Suc-Ala-Glu-Pro-Phe-pNA as substrate. (d) Comparison of serum stability of peptides 1 and 37.

[0208] Figure 38 shows the cellular activity of peptide 37. (a) Cellular uptake of peptides 1, 37 and 46 (5 μM) by HeLa cells analyzed by flow cytometry. MFI, mean fluorescence intensity; None, untreated cells (no peptide). (b) Antiproliferative effects of peptides 37, 46 and 47 on HeLa cells measured by MTT assay. (c) Western blot showing the effect of peptides 1, 37 and 47 on PML protein levels in HeLa cells. β-actin was used as a loading control. (d) Quantification of Western blot results from (c). The reported data are after background subtraction and represent the mean ± SD from 3 independent experiments.

[0209] Details

[0210] The compounds, compositions, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples and Figures included therein.

[0211] Before disclosing and describing the compounds, compositions and methods of the present invention, it should be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, and thus can certainly be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific aspects and is not intended to be limiting.

[0212] In addition, in this specification, various publications are cited. The disclosures of these publications are incorporated by reference in their entirety into this application in order to more fully describe the state of the art to which the disclosed content belongs. The disclosed references are also individually and specifically incorporated by reference into the materials contained herein, which are discussed in the sentences that rely on the references.

[0213] General Definition

[0214] Throughout this specification and the appended claims, reference will be made to a number of terms which shall be defined to have the following meanings.

[0215] Throughout the description and claims of the present application, the word "comprise" and other forms of the word such as "include" and "comprising" mean including but not limited to, and are not intended to exclude, for example, other additives, components, integers or steps.

[0216] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the component" includes mixtures of two or more such components, etc.

[0217] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0218] Ranges can be expressed herein as from "about" a particular value, and / or to "about" another particular value. "About" means within 5% of the value, such as within 4%, 3%, 2% or 1% of the value. When such a range is expressed, another aspect includes from a particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the particular value forms another aspect. It should also be understood that the endpoints of each range are important relative to the other endpoint and independently of the other endpoint.

[0219] As used herein, "subject" refers to an individual. Therefore, "subject" may include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), experimental animals (e.g., mice, rabbits, guinea pigs, etc.), and birds. "Subject" may also include mammals, such as primates or humans. Therefore, the subject may be a human or animal patient. The term "patient" refers to a subject under treatment by a clinician (e.g., a doctor).

[0220] The term "inhibit" refers to a reduction in an activity, reaction, condition, disease or other biological parameter. This may include, but is not limited to, complete elimination of an activity, reaction, condition or disease. This may also include, for example, a 10% reduction in an activity, reaction, condition or disease compared to a natural or control level. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any reduction therebetween compared to a natural or control level.

[0221] "Reduce" or other forms of the word, such as "reduced" or "mitigated" refers to a decrease in an event or characteristic (e.g., tumor growth). It should be understood that this is usually related to some standard or expected value, in other words, it is relative, but does not always require reference to a standard or relative value. For example, "reduce tumor growth" means that the rate of tumor growth is reduced relative to a standard or control.

[0222] "Prevention" or other forms of the word such as "prevent" or "prevent" refers to stopping a particular event or feature, stabilizing or delaying the development or progression of a particular event or feature, or minimizing the chance of a particular event or feature occurring. Preventing does not need to be compared to a control because it is usually more absolute than, for example, reducing. As used herein, certain things can be reduced but not prevented, while certain things that can be reduced can also be prevented. Similarly, certain things can be prevented but not reduced, but certain things that can be prevented can also be reduced. It should be understood that in the case of using reduce or prevent, unless otherwise explicitly stated, the use of other words is also explicitly disclosed. For example, the term "prevention" or "inhibition" can refer to a treatment that prevents or slows the onset of a disease or condition or reduces the severity of a disease or condition. Therefore, if a treatment can treat a disease in a subject with symptoms of the disease, it can also prevent or inhibit a disease in a subject who has not yet suffered some or all of the symptoms.

[0223] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to the improvement of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed to removing the cause of the relevant disease, pathological condition or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition or disorder; preventive treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to supplement another specific treatment directed to the improvement of the relevant disease, pathological condition or disorder.

[0224] The term "anti-cancer" refers to the ability to treat or control cell proliferation and / or tumor growth at any concentration.

[0225] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or condition. Such improvement only requires reduction or change, but not necessarily elimination.

[0226] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0227] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or promotes the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of a compound or composition for 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 in a subject.

[0228] The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked through the carboxyl group of one amino acid to the alpha amino group of another amino acid.

[0229] Unless stated to the contrary, chemical formulas with bonds shown only as solid lines rather than wedges or dashes encompass every possible isomer, such as each enantiomer, diastereomer, and meso compound, as well as mixtures of isomers, such as racemic or scalemic mixtures.

[0230] 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.

[0231] Compound

[0232] Disclosed herein are compounds having activity as cell penetrating peptides. In some examples, the compound may comprise a cell penetrating peptide portion and a cargo portion. The cargo portion may comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof.

[0233] In some instances, the cell penetrating peptide portion is cyclic. In some instances, the cell penetrating peptide portion and the cargo portion are cyclic together. In some instances, the cell penetrating peptide portion is cyclic, and the cargo portion is attached to the cyclic cell penetrating peptide portion structure. In some instances, the cargo portion is cyclic, and the cell penetrating peptide portion is cyclic, and together they form a fused bicyclic system.

[0234] The cell penetrating peptide portion may comprise five or more, more specifically six or more, such as six to twelve, or six to nine amino acids. When there are six to nine amino acids, the compound may have Formula I:

[0235]

[0236] Among them, AA 1 AA 2 AA 3 AA 4 AA 5 AA 6 AA7 ,AA 8 and AA 9 (i.e., AA 1 -AA 9 ) are each independently an amino acid; and m, n and p are independently selected from 0 and 1. When there are more than 9 amino acids, in Formula I, m and p are both 1, and n may be 2 or greater, such as 2 to 10 or 2 to 5. In some examples, three or more amino acids are arginine, and one or more are phenylalanine. In other examples, one or more amino acids are naphthylalanine or tryptophan.

[0237] In some examples, the compound can have Formula I:

[0238]

[0239] Among them, AA 1 AA 2 AA 3 AA 4 AA 5 AA 6 AA 7 ,AA 8 and AA 9 (i.e., AA 1 -AA 9 ) are each independently an amino acid; and m, n and p are independently selected from 0 and 1.

[0240] In some examples, the cell penetrating peptide portion is cyclic, and the compound can be a compound of Formula Ia:

[0241]

[0242] Among them, AA 1 -AA 9 , m, n and p are as defined in Formula I, and wherein the curve represents a covalent bond. The curve can be a covalent bond in the peptide backbone (i.e., a carboxylic acid of one AA forms an amide bond with the α-amine of another AA), a bond between the side chains of two AAs, a bond from one side chain of an AA to the backbone carboxylic acid or α-amine of another AA, or a disulfide bond between two AAs.

[0243] In some examples, the compound further comprises a cargo moiety, and the compound can have Formula II:

[0244]

[0245] wherein the cargo moiety may comprise a detectable moiety, a therapeutic moiety, a targeting moiety, or a combination thereof, and AA 1 -AA 9, m, n and p are as defined in Formula I.

[0246] In some examples, the cell penetrating peptide portion and the cargo portion together are cyclic, and the compound has Formula IIa:

[0247]

[0248] wherein the cargo moiety is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0249] In some examples, the cell penetrating peptide moiety is cyclic, and the cargo moiety is attached to the cyclic cell penetrating peptide moiety structure, and the compound has Formula IIb:

[0250]

[0251] Wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0252] In some examples, the cargo moiety is cyclic and the cell penetrating peptide moiety is cyclic, the cargo moiety and the cell penetrating peptide moiety together form a fused bicyclic ring system, and the compound has Formula IIc:

[0253]

[0254] Wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0255] Cell Penetrating Peptides

[0256] The cell penetrating peptide portion comprises at least 5, more specifically at least 6 amino acids, even more specifically 6 to 12, 6 to 9, 6 to 7, 7 to 8, 8 to 9 and more specifically 6, 7, 8 or 9 amino acids. For the endocyclic motif, at least 5 amino acids can be used. It is also disclosed herein that for the endocyclic structure, some amino acids in the penetrating peptide portion can also be part of the cargo portion. For example, the peptide penetrating portion FNalRR can be formed by FNal and a cargo portion with two Args. In this case, the two Arg residues perform dual functions. Therefore, in some cases, when referring to the peptide penetrating portion, the sequence of the cargo portion is considered.

[0257] For the exocyclic motif, at least 6 amino acids can be used, for example, glutamine can be used to link the cargo.

[0258] Each amino acid can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound as a natural amino acid homologue, which has a structure similar to that of a natural amino acid, so that it simulates 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 20 common natural amino acids or rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. 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 or combinations thereof. These and other and their abbreviations used in this article are listed in Table 1.

[0259] Table 1. Amino acid abbreviations

[0260]

[0261]

[0262] *Single letter abbreviations: When shown herein in upper case, they represent the L-amino acid form, and when shown herein in lower case, they represent the D-amino acid form

[0263] The amino acid can be coupled via a peptide bond.The amino acid can be coupled to the cargo moiety at the amino group, the carboxylate group or the side chain.

[0264] In some examples of Formula I, at least one amino acid comprises naphthylalanine or tryptophan, or an analog or derivative thereof. In some examples of Formula I, at least three amino acids independently comprise arginine, or an analog or derivative thereof. In some examples of Formula I, at least one amino acid comprises phenylalanine, phenylglycine, or histidine, or an analog or derivative thereof. In some examples of Formula I, at least one amino acid comprises glutamine, or an analog or derivative thereof.

[0265] In some examples, the cell penetrating peptide (CPP) portion can be any sequence listed in Table 2. In some examples, the cell penetrating peptide can be the reverse sequence of any sequence listed in Table 2. In some examples, the cell penetrating peptide sequence can be a cyclic form of any sequence listed in Table 2.

[0266] Table 2. CPP sequences - linear or circular

[0267]

[0268]

[0269] Φ = L-naphthylalanine; φ = D-naphthylalanine; Ω = L-norleucine

[0270] In some examples, the cell penetrating peptide portion may be any one of SEQ ID NO: 1 to SEQ ID NO: 29. In some examples, the cell penetrating peptide portion may be a variant of any one of SEQ ID NO: 1 to SEQ ID NO: 29. Peptide variants are well known to those skilled in the art and may involve amino acid sequence modifications. For example, amino acid sequence modifications generally fall into one or more of three categories: substitution, insertion or deletion variants. Insertions include amino and / or carboxyl terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions are generally smaller insertions than amino or carboxyl terminal fusions, for example, about 1 to 3 residues. Deletions are characterized by the removal of one or more amino acid residues from the peptide sequence. Generally, no more than 1 to 3 residues are deleted at any one site in the peptide. Amino acid substitutions are generally single residues, but may occur simultaneously at multiple different positions; insertions are generally about 1 to 3 amino acid residues; and deletions are about 1 to 3 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., 2 residues are deleted or 2 residues are inserted. Substitutions, deletions, insertions, or any combination thereof may be combined to obtain the final construct. Substitution variants are variants in which at least one residue has been removed and a different residue inserted in its place. Such substitutions are generally made according to Table 3 below and are referred to as conservative substitutions.

[0271] Table 3. Amino acid substitutions

[0272]

[0273] Substantial changes in function are made by selecting substitutions that are less conservative than those in Table 3, i.e., by selecting residues that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of ​​the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions generally expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, such as seryl or threonyl, is replaced (or is replaced) with a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is replaced (or is replaced) with any other residue; (c) a residue with a positively charged side chain, such as lysyl, arginyl, or histidyl, is replaced (or is replaced) with an electronegative residue, such as glutamyl or aspartyl; or (d) a residue with a bulky side chain, such as phenylalanine, is replaced (or is replaced) with a residue without a side chain, such as glycine, in which case (e) by increasing the number of sites of sulfation and / or glycosylation.

[0274] For example, replacing one amino acid residue with another amino acid residue that is biologically and / or chemically similar is a conservative substitution known to those skilled in the art. For example, a conservative substitution replaces one hydrophobic residue with another hydrophobic residue, or replaces one polar residue with another polar residue. The substitution includes combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each explicitly disclosed sequence are included in the peptides provided herein.

[0275] It should be understood that one way to define variants of the disclosed cell penetrating peptide moieties is by defining variants based on homology / identity to specific known sequences. For example, SEQ ID NO: 1 to SEQ ID NO: 29 all give specific sequences. Variants of these peptides are specifically disclosed, which have at least 85%, 90%, 95% or 97% homology to SEQ ID NO: 1 to SEQ ID NO: 29. Those skilled in the art will readily understand how to determine the homology of two proteins. For example, homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0276] In addition to variants of SEQ ID NO: 1 to SEQ ID NO: 29, derivatives of these peptides also work in the disclosed methods and compositions. Derivatives are formed by replacing one or more residues with modified residues, wherein the side chains of the residues have been modified. Additional examples are shown in Tables 6 and 18 and include variants thereof.

[0277] Cargo section

[0278] The cargo moiety can include any cargo of interest, such as a linker moiety, a detectable moiety, a therapeutic moiety, a targeting moiety, etc., or any combination thereof. In some examples, the cargo moiety can include one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., a bifunctional linker LC-SMCC); coenzyme A; phospho-coumarylaminopropionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L-2,3-diaminopropionic acid (Dap or J); L-β-naphthylalanine; L-piperidinic 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.

[0279] In some examples, the cargo portion may include any of those listed in Table 4 or derivatives or combinations thereof.

[0280] Table 4. Example cargo parts

[0281] SEQ ID NO abbreviation sequence* 30 <![CDATA[R5]]> RRRRR 31 <![CDATA[A5]]> AAAAA 32 <![CDATA[F4]]> FFFF 33 PCP DE(pCAP)LI 34 <![CDATA[A7]]> AAAAAAA 35 RARAR 36 DADAD 37 DΩUD 38 UTRV

[0282] *pCAP, phosphonocoumarylaminopropionic acid; Ω, norleucine; U, 2-aminobutyric acid.

[0283] Detectable part

[0284] The detectable portion 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 detectable label is not added with other reagents.

[0285] 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 "biocompatible" generally refer to compounds that are generally non-toxic to cells and tissues, along with any of their metabolites or degradation products, and do not cause any significant adverse effects on cells and tissues when incubated (e.g., cultured) in their presence.

[0286] The detectable moiety may comprise 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, perylene diimide, pyrene; azo dyes; xanthene dyes; boron dipyrromethene, aza-boron dipyrromethene, cyanine dyes, metal-ligand complexes such as bipyridine, bipyridyl, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; aza-annulene, squaryl cyanine; 8-hydroxyquinoline, polymethine, luminescence-producing nanoparticles such as quantum dots, nanocrystals; quinolones, terbium complexes; inorganic fluorophores; ion carriers such as crown ethers attached or derivatized 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 tetrabenzomorphine; 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- 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; calcein; bromodimethylol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromophenolphthalein; Congo red; fluorescein; eosin; 2',7'-dichlorofluorescein; 5(6)-carboxy-fluorescein; carboxynaphthofluorescein; 8-hydroxystyrene-1,3,6-trisulfonic acid; heminaphthodifluoride; heminaphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline)dichlororuthenium(II); (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron; platinum(II) octaethylporphyrin; dialkylcarbocyanine; dioctadecylcyclodicarbocyanine; fluorenylmethoxycarbonyl chloride; 7-amino-4-methylcoumarin (Amc); green fluorescent protein (GFP) and derivatives or combinations thereof.

[0287] In some examples, the detectable moiety can include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), or a derivative or combination thereof.

[0288] The detectable moiety can be attached to the cell penetrating peptide moiety at an amino group, a carboxylate group, or a side chain of any amino acid in the cell penetrating peptide moiety (e.g., at an amino group, a carboxylate group, or a side chain or AA 1 -AA xany one of ).

[0289] Treatment

[0290] The disclosed compounds may also comprise a therapeutic moiety. In some instances, the cargo moiety comprises a 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.

[0291] Therapeutic moieties may include a variety of drugs, including antagonists such as enzyme inhibitors, agonists such as transcription factors that result in increased expression of a desired gene product (although antagonistic transcription factors may also be used as will be appreciated by those skilled in the art). In addition, therapeutic moieties include those agents that are capable of directing toxicity and / or capable of inducing toxicity to healthy and / or unhealthy cells in the body. Furthermore, therapeutic moieties are capable of inducing and / or priming the immune system against potential pathogens.

[0292] The therapeutic moiety can, for example, comprise 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.

[0293] The therapeutic moiety may include an anticancer agent. Examples of anticancer agents include 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, isotretinoin (Accutane), actinomycin D, doxorubicin, Adrucil, Anguining, Ala-Cort, aldesleukin, alemtuzumab, alitretinoin, Alkaban-AQ, Ai Kelan, all-trans retinoic acid, alpha interferon, hexamethylmelamine, methotrexate, amifostine, glutethimide, anagrelide, andra, anastrozole, cytarabine, Anran Aisipu, Acorda, Arimidex, Arno, arsenic trioxide, asparaginase, ATRA, Avastin, BCG, BCNU, bevacizumab, bexarotene, bicalutamide, BiCNU, Blenoxane, bleomycin, bortezomib, busulfan, Busulfex, C225, leucovorin, Campath, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine Tablets, Casodex, CCNU, CDDP, CeeNU, Zhengdingmycin, Cetuximab, Chlorambucil, Cisplatin, Folinic Acid Factor, Cladribine, Cortisone, Kemeijing, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposome, Cytosar-U, Cytoxan, Dacarbazine, Actinomycin, Dapoxetine alpha, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, DaunoXome, Decadron, Delta-Cortef, Prednisone, Denileukin-toxin conjugate, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Adriamycin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralon, Efudex, Elica, Allens, Eloxatin, Esba, Emcyt, Epirubicin, Epoetin alpha, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Fanbifu, Etoposide, Etoposide phosphate, Eulexin, Evita, Exemestane, Falutong, Falod, Furulina, Filgrastim, Floxuridine, Fudahua, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Fluoramide, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemzar, Gleevec, Leucine acetateProrelin, Lupron Depot, Matulane, Maxidex, Nitrogen mustard, Nitrogen mustard hydrochloride, Medrol, Methylprednisolone, Megace, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumiga, Eubogen, Nilandron, Nilutamide, Nitrogen mustard, Novaldex, Novaldex, Novoxolin, Octreotide, Octreotide acetate, Oncospar, Vincristine, Ontak, Onxal, Opprevirgin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronic acid, Panretin, Baldine, Pediapred, PEG interferon, pegaspargase, pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, phenylalanine mustard, Platinol, Platinol-AQ, prednisolone, prednisone, Prelone, procarbazine, PROCRIT, Proliferin, Prolifeprospan 20 with carmustine implant, Purinethol, raloxifene, amoxazol, rituximab, Roveron-A (interferon alpha-2a), Rubex, rubeclocycline hydrochloride, Sandostatin, Sandostatin LAR, Sargamodin, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Tacozole, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalidomide, TheraCys, Thioguanine, Thioguanine tablets, Thiophosphamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Retinoic acid, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Vanbisilane, Vansanone, Viadur, Vinblastine, Vinblastine sulfate, Vincasar Pfs, vincristine, vinorelbine, vinorelbine tartrate, VLB, VP-16, Viagra, Xeloda, Zanosar, Zevalin, Zinecard, Noradrenaline, Zoledronic acid, Zometa, Glider chip, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, fluoxymesterone, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hemeixin, Hydrea, hydrocortisone acetate, hydrocortisone,Hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone phosphate, hydroxyurea, isobetumomab, tentan isobetumomab, edramycin, 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, leukine, leuprorelin, vincastin, lestatin, liposomal Ara-C, Liquid Pred, lomustine, L-PAM, L-sabolin, Meticorten, mitomycin, mitomycin-C, mitoxantrone, M-prednisone, MTC, MTX, nitrogen mustard, mitomycin, Myleran, Iressa, Elanodiken, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. The therapeutic moiety may also comprise a biopharmaceutical, such as an antibody.

[0294] In some examples, the therapeutic moiety can include an antiviral agent, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), and the like.

[0295] In some examples, the therapeutic moiety can include an antibacterial agent such as acaprolone, sodium acetylsulfone, alamecin, alexidine, azepine, pivmecillin, amicycline, amlofloxacin, amlofloxacin mesylate, amikacin, amikacin sulfate, aminosalicylic acid, sodium aminosalicylate, amoxicillin, amphotericin, ampicillin, ampicillin sodium, apacillin sodium, apramycin, aspartic acid, aspartic acid sulfate, avermectin, avoparcin, azithromycin, azlocillin, azlocillin sodium, bacampicillin hydrochloride, bacitracin, bacitracin methylene disalicylate, bacitracin zinc, bambemycin, Calcium benzoyl, erythromycin sulfate, betamycin sulfate, biapenem, benimycin, phensalamide hydrochloride, magnesium sulfate pyridamole, butikacin, butyryl sulfate, capreomycin sulfate, carbadox, carbenicillin disodium, carbenicillin indenyl sodium, carbenicillin phenyl sodium, carbenicillin potassium, coumarin sodium, cefaclor, cefdroxil, cefmandole, cefmandole sodium, cefmandole sodium, cefoperazone, ceftriaxone, cefazolin sodium, cefazolin, cefazolin sodium, cefoperazone, Cefdinir, Cefepime, Cefepime Hydrochloride, Cefotaxime, Cefixime, Cefmenoxime Hydrochloride, Cefmetazole, Cefmetazole Sodium, Cefonicid Monosodium, Cefonicid Sodium, Cefoperazone Sodium, Cefdinir, Ceftriaxone Sodium, Cefotetan, Cefotetan Disodium, Ceftiam Hydrochloride, Cefoxitin, Cefoxitin Sodium, Cefimazole, Cefoperazone Sodium, Cefpiramide, Cefpiramide Sodium, Cefpiramide Sulfate, Cefpodoxime Propyl Axetil, Cefprozil, Cefuroxime Sodium, Ceftazidime, Ceftibuten, Cefuroxime sodium, ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroximepixetil, ceftriaxone sodium, cephalexin, cephalexin hydrochloride, cephalosporin, ceftriaxone, ceftriaxone sodium, cefpirin sodium, cephaloridine, cetocycline hydrochloride hydrochloride), acetyl chloramphenicol, chloramphenicol, chloramphenicol palmitate, chloramphenicol pantothenate complex, chloramphenicol sodium succinate, chlorhexidine aminophenylphosphate, chloroxylenol, chlortetracycline hydrogen sulfate, chlortetracycline hydrochloride, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, cilomycin, clarithromycin, clinafloxacin hydrochloride, clindamycin, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, clofazimine, benzathine cloxacillin, cloxacillin sodium, cloxacillin (cloxyquin), colistin sodium mesylate, coumermycin, coumermycin sodium, cyclocillin, cycloserine, dalfopristin, dapsone, daptomycin, demeclocycline, demeclocycline hydrochloride, demeclocycline, dinomycin, dicloxacillin, dicloxacillin sodium, dihydrostreptomycin sulfate, dipyridinethione, dirithromycin, doxycycline, Doxycycline calcium, doxycycline phosphate complex, doxycycline hydrochloride, drofloxacin sodium, enoxacinfloxacin, epicillin, epitetracycline hydrochloride, erythromycin, erythromycin acetate stearate, erythromycin estolate, erythromycin ethylsuccinate, erythromycin glucoheptonate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, ethambutol hydrochloride, ethionamide, fleroxacin, flucloxacillin, flutrithymidine, flumequine, fosfomycin, fosfomycin tromethamine, furomocillin, furanium chloride, furanium tartrate, sodium fusidate, fusidic acid, gentamicin sulfate, glomonam, gramicidin, halogenated propynyloxybenzene, hetacillin, hetacillin potassium, hexedine, ibafacin, imipenem, isoconazole, isopamicin, isoniazid, Josamycin, kanamycin sulfate, kedamycin, levofloxacin, levopropylcillin potassium, erythromycin, lincomycin, lincomycin hydrochloride, lomefloxacin, lomefloxacin hydrochloride, lomefloxacin mesylate, loracarbef, mafenide, meclocycline, meclocycline sulfosalicylate, mequidox, meropenem, methacycline, methacycline hydrochloride, Methenamine, methenamine hippurate, methenamine mandelate, methicillin sodium, metioprim, metronidazole hydrochloride, metronidazole phosphate, mezlocillin, mezlocillin sodium, minocycline, minocycline hydrochloride, pyrimycin hydrochloride, monensin, monensin sodium, nafcillin sodium, nalidixic acid sodium, nalidixic acid, nattomycin, nibumycin, neomycin palmitate, neomycin sulfate, Neomycin undecylenate, Netilmicin sulfate, Neutramycin, Nitrofurazol, Nifuroxene, Nifuroxine, Nifuratel, Nifuron, Nifurdazi, Nifuramide, Nifupirol, Nifuquinazole, Nitrothiazole, Nitrocycline, Nitrofurantoin, Nitroamide, Norfloxacin, Neomycin Sodium, Ofloxacin, Onatoprene, Oxacillin, Oxacillin Sodium, Oxime, Oxime Sodium, Oxalic Acid, Oxytetracycline, Oxytetracycline Calcium, Oxytetracycline Hydrochloride, Parimicin, Parachlorophenol, Paolomycin, Pefloxacin, Pefloxacin Mesylate, Penicillin, Penicillin G Benzathine, Penicillin G Potassium, Penicillin G procaine, penicillin G sodium, penicillin V, penicillin V benzathine, hyrobacillin V, penicillin V potassium, tebuconazole sodium, phenylaminosalicylate / ester, piperacillin sodium, picolinicillin sodium, picolinicillin sodium, prilocybin hydrochloride, pivampicillin hydrochloride, pivampicillin pamoate, pivampicillin propionate, polymyxin B sulfate, methyl mitomycin (porfiromycin), prupicacin, pyrazinamide, zinc pyrithione, quinethamide acetate, quinuclamine acetate, quinupristin, racemic thiamphenicol, ramoplanin, ranimycin, relomycin, rapamycin, rifabutin, riformestane, rifaxilan, rifaamide, rifampicin, rifapentine, rifapentineFuximin, Rolicycline, Rolicycline nitrate, Roxamicin, Roxamicin butyrate, Roxamicin propionate, Roxamicin sodium phosphate, Roxamicin stearate, Rosofloxacin, Roxarsone, Roxithromycin, Tricycline, Triterpenoid Sodium, Samoxicillin, Samoxicillin, Sescofungin, Sisomicin, Sisomicin Sulfate, Sparfloxacin, Spectinomycin Hydrochloride, Spiramycin, Stallimycin Hydrochloride, Cetirycin, Streptomycin Sulfate, Streptomycin Nicotinamide, Sulfadiazine, Sulfadiazine, Sulfadiazine, Sulfacetamide, Sulfacetamide Sodium, Sulfoxetine, Sulfadiazine, Sulfadiazine Sodium, Sulfadoxine, Sulfadiazine, Sulfamerazine, Sulfadiaz ... Sulfadimethoxine, zinc sulfadimethoxine, sulfanitrophenyl, sulfasalazine, sulfisothiazole, sulfathiazole, sulfapyrazole, sulfisoxazole, acetylthioisoxazole, sulfisoxazole diethanolamine, sulfocin, sulfopenem, sultamicillin, sodium sulfamethoxazole, thiacillin hydrochloride, teicoplanin, temafloxacin hydrochloride, temocillin tetracycline, tetracycline hydrochloride, tetracycline phosphate complex, alloxan, thiamphenicol, thifencillin potassium, ticarcillin cresyl sodium, ticarcillin disodium, ticarcillin monosodium, ticarcillinone, clobenzimidol chloride, tobramycin, tobramycin sulfate, tofloxacin, trimethoprim, trimethoprim sulfate, trisulfuron, troleandomycin, spectinomycin sulfate, gramicidin, Vancomycin, vancomycin hydrochloride, vilamicin or zorbamycin.

[0296] In some instances, the therapeutic moiety can comprise an anti-inflammatory agent.

[0297] In some instances, the therapeutic moiety can comprise dexamethasone (Dex).

[0298] In other examples, the therapeutic moiety comprises a therapeutic protein. For example, some human enzymes (e.g., lysosomal storage diseases) are deficient. Disclosed herein is the delivery of these enzymes / proteins to human cells by linking them to one of the disclosed cell penetrating peptides. The disclosed cell penetrating peptides have been tested with proteins (e.g., GFP, PTP1B, actin, calmodulin, troponin C) and shown to be effective.

[0299] In some instances, the treatment moiety comprises a targeting moiety. The targeting moiety can comprise, for example, an amino acid sequence that can target one or more enzyme domains. In some instances, the targeting moiety can comprise an inhibitor of an enzyme that can work in a disease such as cancer, cystic fibrosis, diabetes, obesity or its combination. For example, the targeting moiety can comprise any sequence listed in the table 5.

[0300] Table 5. Examples of targeting moieties

[0301]

[0302]

[0303] *Fpa, Σ: L-4-fluorophenylalanine; Pip, Θ: L-homoproline; Nle, Ω: L-norleucine; Phg, ΨL-phenylglycine; F2Pmp, Λ: L-4-(phosphonodifluoromethyl)phenylalanine; Dap, L-2,3-diaminopropionic acid; Nal, Φ': L-β-naphthylalanine; Pp, θ: L-piperidinic acid; Sar, Ξ: sarcosine; Tm, trimesic acid.

[0304] The targeting moiety and the cell penetrating peptide moiety may overlap, ie residues forming the cell penetrating peptide moiety may also be part of the sequence forming the targeting moiety and vice versa.

[0305] The therapeutic moiety can be attached to the cell penetrating peptide moiety at an amino group, a carboxylate group, or a side chain of any amino acid in the cell penetrating peptide moiety (e.g., at an amino group, a carboxylate group, or a side chain or AA 1 -AA x In some instances, the therapeutic moiety can be linked to a detectable moiety.

[0306] In some examples, the therapeutic moiety can comprise a targeting moiety that can act as an inhibitor against Ras (eg, K-Ras), PTP1B, Pin1, Grb2SH2, CAL PDZ, etc., or a combination thereof.

[0307] Ras is a protein that in humans is encoded by the RAS gene. Normal Ras proteins play an essential role in normal tissue signaling, and mutations in the Ras gene have been implicated in the development of many cancers. Ras can be used as a molecular on / off switch, and once it is turned on, Ras recruits and activates proteins that are essential for the propagation of growth factor and other receptor signals. Mutated forms of Ras have been implicated in a variety of cancers, including lung cancer, colon cancer, pancreatic cancer, and various leukemias.

[0308] 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 is also involved in the development of breast cancer.

[0309] Pin1 is an enzyme that binds a subset of proteins and acts as a post-phosphorylation control in regulating protein function. Pin1 activity can modulate the outcome of proline-directed kinase signaling, which can regulate cell proliferation and cell survival. Dysregulation of Pin1 can play a role in various diseases. Upregulation of Pin1 may be involved in certain cancers, and downregulation of Pin1 may be involved in Alzheimer's disease. Pin1 inhibitors may have therapeutic implications for cancer and immune diseases.

[0310] Grb2 is an adaptor protein involved in signal transduction and cell communication. The Grb2 protein contains an SH2 domain that can bind 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 can block transformation and proliferation of various cell types.

[0311] Recently reported, 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, which is 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). Thus, disclosed herein is a method of treating a subject with cystic fibrosis by administering an effective amount of a compound or composition disclosed herein. The compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can be used as an inhibitor against CAL PDZ. In addition, the compound or composition disclosed herein may be administered together with a molecule that corrects CFTR function.

[0312] Specific Examples

[0313] In some examples, the compound can have Formula I:

[0314]

[0315] Among them, AA 1 AA 2 AA 3 AA 4 AA 5 AA6 AA 7 , A.A. 8 and AA 9 (i.e., AA 1 -AA 9 ) are each independently an amino acid; and m, n and p are independently selected from 0 and 1.

[0316] In some examples of Formula I, m, n, and p are 0, and the compound has Formula I-1:

[0317] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6

[0318] I-1

[0319] Among them, AA 1 -AA 6 As defined in Formula I.

[0320] In some examples of Formula I, m is 1, n and p are 0, and the compound has Formula I-2:

[0321] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7

[0322] I-2

[0323] Among them, AA 1 -AA 7 As defined in Formula I.

[0324] In some examples of Formula I, m and n are 1, p is 0, and the compound has Formula I-3:

[0325] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8

[0326] I-3

[0327] Among them, AA 1 -AA 8 As defined in Formula I.

[0328] In some examples of Formula I, m, n, and p are 1, and the compound has Formula I-4:

[0329] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -AA 9

[0330] I-4

[0331] Among them, AA 1 -AA 9 As defined in Formula I.

[0332] In some examples, the cell penetrating peptide portion is cyclic, and the compound can have Formula Ia:

[0333]

[0334] Among them, AA 1 -AA 9 , m, n and p are as defined in Formula I, and wherein the curved line represents a covalent bond.

[0335] In some examples of Formula Ia, m, n, and p are 0, and the compound has Formula Ia-1:

[0336]

[0337] Among them, AA 1 -AA 6 As defined in Formula I.

[0338] In some examples of Formula Ia, m is 1, n and p are 0, and the compound has Formula Ia-2:

[0339]

[0340] Among them, AA 1 -AA 7 As defined in Formula I.

[0341] In some examples of Formula Ia, m and n are 1, p is 0, and the compound has Formula Ia-3:

[0342]

[0343] Among them, AA 1 -AA 8As defined in Formula I.

[0344] In some examples of Formula Ia, m, n, and p are 1, and the compound has Formula Ia-4:

[0345]

[0346] Among them, AA 1 -AA 9 As defined in Formula I.

[0347] In some examples, the compound further comprises a cargo moiety, and the compound can have Formula II:

[0348]

[0349] wherein the cargo moiety may comprise a detectable moiety, a therapeutic moiety, a targeting moiety, or a combination thereof, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0350] In some examples of Formula II, m, n and p are 0, and the compound has Formula II-1:

[0351] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -goods

[0352] II-1

[0353] Among them, AA 1 -AA 6 As defined in Formula I, and the cargo is as defined in Formula II.

[0354] In some examples of Formula II, m is 1, n and p are 0, and the compound has Formula II-2:

[0355] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -goods

[0356] II-2

[0357] Among them, AA 1 -AA 7 As defined in Formula I, and the cargo is as defined in Formula II.

[0358] In some examples of Formula II, m and n are 1, p is 0, and the compound has Formula II-3:

[0359] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -goods

[0360] II-3

[0361] Among them, AA 1 -AA 8 As defined in Formula I, and the cargo is as defined in Formula II.

[0362] In some examples of Formula II, m, n and p are 1, and the compound has Formula II-4:

[0363] AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -AA 9 -goods

[0364] lI-4

[0365] Among them, AA 1 -AA 9 As defined in Formula I, and the cargo is as defined in Formula II.

[0366] In some examples, the cell penetrating peptide portion and the cargo portion together are cyclic, and the compound has Formula IIa:

[0367]

[0368] wherein the cargo moiety is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0369] In some examples of Formula IIa, m, n and p are 0, and the compound has Formula IIa-1:

[0370]

[0371] Among them, AA 1 -AA6 As defined in Formula I, and the cargo is as defined in Formula II. Also disclosed herein is Formula IIa-1, wherein one of AA1-AA6 is absent (ie, there are 5 amino acids in the internal ring structure).

[0372] In some examples of Formula IIa, m is 1, n and p are 0, and the compound has Formula IIa-2:

[0373]

[0374] Among them, AA 1 -AA 7 As defined in Formula I, and the cargo is as defined in Formula II.

[0375] In some examples of Formula IIa, m and n are 1, p is 0, and the compound has Formula IIa-3:

[0376]

[0377]

[0378] Among them, AA 1 -AA 8 As defined in Formula I, and the cargo is as defined in Formula II.

[0379] In some examples of Formula IIa, m, n and p are 1, and the compound has Formula IIa-4:

[0380]

[0381] Among them, AA 1 -AA 9 As defined in Formula I, and the cargo is as defined in Formula II.

[0382] In some examples, the cell penetrating peptide moiety is cyclic, and the cargo moiety is attached to the cyclic cell penetrating peptide moiety structure, and the compound has Formula IIb:

[0383]

[0384] Wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0385] In some examples of Formula IIb, m, n and p are 0, and the compound has Formula IIb-1:

[0386]

[0387] Among them, AA 1 -AA 6 As defined in Formula I, and the cargo is as defined in Formula II.

[0388] In some examples of Formula IIb, m is 1, n and p are 0, and the compound has Formula IIb-2:

[0389]

[0390] Among them, AA 1 -AA 7 As defined in Formula I, and the cargo is as defined in Formula II.

[0391] In some examples of Formula IIb, m and n are 1, p is 0, and the compound has Formula IIb-3:

[0392]

[0393] Among them, AA 1 -AA 8 As defined in Formula I, and the cargo is as defined in Formula II.

[0394] In some examples of Formula IIb, m, n and p are 1, and the compound has Formula IIb-4:

[0395]

[0396] Among them, AA 1 -AA 9 As defined in Formula I, and the cargo is as defined in Formula II.

[0397] In some examples, the cargo moiety is cyclic and the cell penetrating peptide moiety is cyclic, the cargo moiety and the cell penetrating peptide moiety together form a fused bicyclic ring system, and the compound has Formula IIc:

[0398]

[0399] Wherein the cargo portion is as defined in Formula II, and AA 1 -AA 9 , m, n and p are as defined in Formula I.

[0400] In some examples of Formula IIc, m, n and p are 0, and the compound has Formula IIc-1:

[0401]

[0402] Among them, AA 1 -AA 6As defined in Formula I, and the cargo is as defined in Formula II.

[0403] In some examples of Formula IIc, m is 1, n and p are 0, and the compound has Formula IIc-2:

[0404]

[0405] Among them, AA 1 -AA 7 As defined in Formula I, and the cargo is as defined in Formula II.

[0406] In some examples of Formula IIc, m and n are 1, p is 0, and the compound has Formula IIc-3:

[0407]

[0408] Among them, AA 1 -AA 8 As defined in Formula I, and the cargo is as defined in Formula II.

[0409] In some examples of Formula IIc, m, n and p are 1, and the compound has Formula IIc-4:

[0410]

[0411] Among them, AA 1 -AA 9 As defined in Formula I, and the cargo is as defined in Formula II.

[0412] In some examples, the compound can include any of the compounds in Table 6. Other examples are shown in Table 18 below.

[0413] Table 6. Example compounds

[0414]

[0415]

[0416]

[0417]

[0418] *Fpa, Σ: L-4-fluorophenylalanine; Pip, Θ: L-homoproline; Nle, Ω: L-norleucine; Phg, ΨL-phenylglycine; F2Pmp, Λ: L-4-(phosphonodifluoromethyl)phenylalanine; Dap, J: L-2,3-diaminopropionic acid; Nal, Φ': L-β-naphthylalanine; Pp, θ: L-piperidinic acid; Sar, Ξ: sarcosine; Tm, trimesic acid; Φ, L-2-naphthylalanine; Rho, rhodamine B; Dex, dexamethasone; FITC, fluorescein isothiocyanate; miniPEG, 8-amino-3,6-dioxaoctanoic acid; pCAP, phosphocoumarylaminopropionic acid; Amc, 7-amino-4-methylcoumarin; FITC, fluorescein isothiocyanate; U, 2-aminobutyric acid.

[0419] Table 7. Previously reported cell penetrating peptides

[0420]

[0421] Also disclosed herein are compositions comprising the compounds described herein.

[0422] 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 the specific substituents found on the compounds. Under conditions where the compounds disclosed herein are sufficiently alkaline or acidic to form stable non-toxic acid or base salts, it may be appropriate to administer the compounds as 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, alpha-ketoglutarate, alpha-sugar phosphates, maleates, toluenesulfonates, and mesylates. Pharmaceutically acceptable salts of compounds can be obtained using standard procedures well known in the art, such as by reacting a sufficiently basic compound (e.g., amine) with a suitable acid providing a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0423] Preparation method

[0424] It will be appreciated by those skilled in the art that the compounds described herein can be prepared by various methods known to those skilled in the art of organic synthesis or variations thereof. 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 these conditions can be determined by those skilled in the art.

[0425] The changes of the compounds described herein include 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 suitable protecting groups can be determined by those skilled in the art. The chemistry 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.

[0426] Starting materials and reagents used to prepare the disclosed compounds and compositions can be obtained 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 according to the procedures set forth 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 Supplements (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.

[0427] The reactions that produce 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 under which the reactions are carried out, i.e., temperature and pressure. The reactions can be carried out in one solvent or in a mixture of more than one solvent. The formation of products or intermediates 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 (eg UV-visible) or mass spectrometry, or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0428] The disclosed compounds can be prepared by solid phase peptide synthesis, wherein the amino acid α-N-terminus is protected by an acid or base protecting group. Such a protecting group should have the property of being stable to the peptide bond forming conditions, and be easy to remove without destroying the growing peptide chain or the racemization of any chiral center contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenyl isopropoxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylthio, 2-cyano-tert-butyloxycarbonyl, etc. For the synthesis of the disclosed compounds, the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred. Other preferred side chain protecting groups are for side chain amino groups such as lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, 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, for cysteine, trityl (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 reactions and that are insoluble in the medium used. The solid support used for the synthesis of the α-C-terminal carboxyl peptides was 4-hydroxymethylphenoxymethyl co-poly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin) obtained from Applied Biosystems (Foster City, Calif). The α-C-terminal amino acid is attached 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)phosphonium hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), in a solvent such as dichloromethane or DMF at a temperature of 10°C to 50°C for about 1 to about 24 hours.When the solid support is a 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, and then coupled with the α-C-terminal amino acid as described above. One method for coupling to the deprotected 4(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is a DMF solution of O-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). The coupling of consecutive protected amino acids can be performed in an automatic peptide synthesizer. In one example, the α-N-terminus of the amino acid 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 in approximately 3-fold molar excess, and the coupling is preferably performed in DMF. The coupling agents 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, which can be performed 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 agent comprising thiophene methyl ether, 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, for example 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 group can be completed using the above-mentioned cleavage mixture. The fully deprotected peptide can be purified by using any or all of the following types of chromatography steps: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography, for example on Sephadex G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), particularly reversed phase HPLC with octyl or octadecylsilane-silica bonded phase column packings.

[0429] How to use

[0430] 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.

[0431] 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 compounds or compositions described herein or a pharmaceutically acceptable salt thereof. The compounds and compositions described herein or a pharmaceutically acceptable salt thereof can be used to treat cancer in humans (e.g., pediatric and elderly populations) and animals (e.g., veterinary applications). The disclosed method may optionally include identifying patients who are or may need to be treated for 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 or tumor. 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 lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, etc.) and lymphomas (Hodgkin and non-Hodgkin lymphoma), and multiple myeloma.

[0432] The methods for treating or preventing cancer described herein may also include treatment with one or more other agents (e.g., anticancer agents or ionizing radiation). One or more other agents as described herein and compounds and compositions or pharmaceutically acceptable salts thereof may be administered in any order, including simultaneous administration, and at intervals of up to several days in time. The method may also include more than a single administration of one or more other agents and / or compounds and compositions or pharmaceutically acceptable salts thereof as described herein. The administration of one or more other 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 other agents, compounds and compositions or pharmaceutically acceptable salts thereof as described herein may be combined into pharmaceutical compositions comprising one or more other agents.

[0433] For example, the compounds or compositions described herein, or pharmaceutically acceptable salts thereof, can be combined with other anticancer agents into pharmaceutical compositions, such as 13-cis-retinoic acid, 2-amino-6-mercaptopurine, 2-CdA, 2-chlorodeoxyadenosine, 5-fluorouracil, 6-thioguanine, 6-mercaptopurine, isotretinoin (Accutane), actinomycin D, doxorubicin, Adrucil, Anguining, Ala-Cort, aldesleukin, alemtuzumab, alitretinoin, Alkaban-AQ, Ai Kelan, all-trans retinoic acid, alpha interferon, hexamethylmelamine, methotrexate, amifostine, glutethimide, anagrelide, andrafenib, anastrozole, cytarabine, Anran Aisipu, Acorda, Arimidex, Arno, arsenic trioxide, asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Folinate, Campath, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine Tablets, Casodex, CCNU, CDDP, CeeNU, Zhengdingmycin, Cetuximab, Chlorambucil, Cisplatin, Folinic Acid Factor, Cladribine, Cortisone, Camejing, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposome, Cytosar-U, Cytoxan, Dacarbazine, Actinomycin, Dapoxetine alpha, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposome, DaunoXome, Decadron, Delta-Cortef, Prednisone, Denileukin-toxin conjugate, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposome, Droxia, DTIC, DTIC-Dome, Dularon, Efudex, Elica, Allens, Lexadin, Esba, Emcyt, Epirubicin, Epoetin alpha, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Fanbifu, Etoposide, Etoposide phosphate, Eulexin, Evita, Exemestane, Falutong, Falod, Furulina, Filgrastim, Floxuridine, Fudahua, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Fluoxetine, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab,Gemzar, Gleevec, Lupron acetate, Lupron Depot, Matulane, Maxidex, Mechlorethamine, Mechlorethamine hydrochloride, Medrol, Methylprednisolone, Megace, Megestrol acetate, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Numiga, Eubojin, Nilandron, Nilutamide, Mechlorethamine, Novaldex, Noxolin, Octreotide, Octreotide acetate, Oncospar, Vincristine, Ontak, Onxal, Opprevirgin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronic acid, Panretin, Baldine, Pediapred, PEG-interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, phenylalanine mustard, Platinol, Platinol-AQ, prednisolone, prednisone, Prelone, Procarbazine, PROCRIT, Prolife, Prospan 20 with carmustine implant, Purinethol, Raloxifene, Amoxan, Rituxan, Rituximab, Roveron-A, Rubex, Rubeclocycline hydrochloride, Sandostatin, Sandostatin LAR, Sagamodin, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Tacozole, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalidomide, TheraCys, Thioguanine, Thioguanine tablets, Thiophosphamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Retinoic acid, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, Vanbisil, Vansan, Viadur, Vinblastine, Vinblastine sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Viagra, Xeloda, Zanosar, Zevalin, Zinecard, Zoledronic acid, Zometa, Glider chip, Glivec, GM-CSF, Goserelin, Granulocyte colony-stimulating factor, Fluoxymesterone, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hemeixin, Hydrorea, Hydrocort acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortisone phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Edemycin, 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, Leukine, Leuprorelin, Venkate, Lestatin, Liposomal Ara-C, Liquid Pred, lomustine, L-PAM, L-sabolin, Meticorten, mitomycin, mitomycin-C, mitoxantrone, M-prednisone, MTC, MTX, nitrogen mustard, mitoxantrone, Myleran, Iressa, Elanodiken, Isotretinoin, Kidrolase, Lanacort, L-asparaginase and LCR. Other anticancer agents may also include biological drugs, such as antibodies.

[0434] 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 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.

[0435] 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. The term ionizing radiation as used herein refers to radiation including particles or photons having sufficient energy or that can generate sufficient energy to produce ionization through nuclear interactions. An example of ionizing radiation is x-radiation. An effective amount of ionizing radiation refers to an ionizing radiation dose 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.

[0436] The methods and compounds described herein can be used for both preventive and therapeutic treatments. The term treatment as used herein includes prevention, delayed onset, alleviation, eradication or delay of the exacerbation of signs or symptoms after onset and prevention of recurrence. For preventive use, a therapeutically effective amount of the compounds and compositions described herein or a pharmaceutically acceptable salt thereof is administered before onset (e.g., before obvious signs of cancer), during early onset (e.g., when initial signs and symptoms of cancer appear), or after the confirmed development of cancer. Preventive administration can occur several days to several years before the manifestation of symptoms of infection. Preventive administration can be used, for example, for chemopreventive treatment of subjects presenting precancerous lesions, subjects diagnosed with early malignancies, and subgroups with susceptibility (e.g., family, race, and / or occupation) to specific cancers. Therapeutic treatment includes administering a therapeutically effective amount of the compounds and compositions described herein or a pharmaceutically acceptable salt thereof to the subject after diagnosing cancer.

[0437] In some examples of methods of 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 can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or a combination thereof.

[0438] The disclosed subject matter also relates to methods for treating subjects with metabolic disorders or diseases. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein are administered to subjects suffering from metabolic diseases and in need of such treatment. In some instances, metabolic disorders may include type II diabetes. In some instances of methods for treating, preventing or improving a subject's metabolic disorder, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can be used as an inhibitor for PTP1B. In a specific instance of the method, the subject is obese, and the method includes treating the subject's obesity by administering a composition disclosed herein.

[0439] The disclosed subject matter also relates to methods for treating subjects with immune disorders or conditions. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject suffering from an immune disorder and in need of such treatment. In some examples of therapeutic methods for treating, preventing or ameliorating an immune disorder in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can be used as an inhibitor against Pin1.

[0440] The disclosed subject matter also relates to methods of 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 such treatment. In some examples of methods of treating cystic fibrosis in a subject, the compound or composition administered to the subject may include a therapeutic moiety, which may include a targeting moiety that can act as an inhibitor against CAL PDZ.

[0441] Compositions, formulations and methods of administration

[0442] The in vivo application of the disclosed compounds and compositions comprising them can be achieved by any suitable method and technology currently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art, including, for example, oral, nasal, rectal, topical and parenteral administration routes. The term parenteral as used herein 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 can be administered continuously or at different intervals as easily determined by those skilled in the art.

[0443] 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.

[0444] 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 readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by EW Martin (1995) describes formulations that can be used in conjunction 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 facilitate the effective administration of the compound. The composition used can also be in a variety of forms. These include, for example, solid, semisolid and liquid dosage forms, such as tablets, pills, powders, liquid solvents 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 includes 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 one or more subject compounds in a total amount of about 0.1% to 100% based on the total weight of the composition including carrier or diluent.

[0445] Preparations suitable for administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The preparation may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, before use, and may be stored under freeze drying (lyophilization) conditions that require only sterile liquid carrier conditions, For example, water for injection. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, etc. It should be understood that, in view of the type of preparation discussed, in addition to the ingredients specifically mentioned above, the compositions disclosed herein may include other agents conventional in the art.

[0446] Compounds disclosed herein and compositions comprising them can be delivered to cells by direct contact with cells or by carrier means. Carrier means for delivering compounds and compositions to cells are known in the art, such as encapsulating the composition in a liposome portion. Another means for delivering compounds disclosed herein and compositions to cells includes connecting 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 No. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow the composition to be transported through a biological membrane. U.S. Application Publication No. 20020035243 also describes a composition for transferring a biological part across a cell membrane 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] (20:80 molar ratio) (as used in GLIADEL); chondroitin; chitin and chitosan.

[0447] For the treatment of tumor disorders, the compounds disclosed herein can be administered in combination with other anti-tumor or anti-cancer substances and / or with radiation and / or photodynamic therapy and / or with surgical treatment to patients in need of treatment to remove tumors. These other substances or treatments can be administered simultaneously with the compounds disclosed herein or not. For example, the compounds disclosed herein can be used in combination with mitotic inhibitors (e.g., paclitaxel or vinblastine), alkylating agents (e.g., cyclophosphamide or ifosfamide), antimetabolites (e.g., 5-fluorouracil or hydroxyurea), DNA intercalators (e.g., doxorubicin or bleomycin), topoisomerase inhibitors (e.g., etoposide or camptothecin), anti-angiogenic agents (e.g., angiostatin), antiestrogens (e.g., tamoxifen) and / or other anti-cancer drugs or antibodies, such as GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc), or immunotherapeutics (e.g., ipilimumab and bortezomib).

[0448] 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 (e.g., tumor sites or benign skin growths, such as by 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 absorbable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly into the food of the patient's diet. For oral therapeutic administration, the active compounds 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.

[0449] 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 substances: 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 flavorings 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 be used as coatings to exist 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. Syrups or elixirs may contain the active compound, sucrose or fructose as sweeteners, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavors. Of course, any material used to prepare any unit dosage form should be pharmaceutically acceptable and substantially nontoxic in the amount used. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0450] 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 active agents or their salts can be prepared in water, optionally mixed with nontoxic surfactants. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof and in oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth.

[0451] The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder containing the active ingredient, which is suitable for the temporary preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The final dosage form should be sterile, mobile and stable under manufacturing and storage conditions. The liquid carrier or carrier can be a solvent or liquid dispersion medium, which includes, for example, water, ethanol, polyols (such as 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 surfactants. 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 sugars, buffers or sodium chloride. The extended absorption of injectable compositions can be achieved by comprising agents that delay absorption (for example, aluminum monostearate and gelatin).

[0452] Sterile injectable solutions are prepared by adding the compounds and / or agents disclosed herein and various other ingredients listed above in the desired amount in an appropriate solvent, 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 ingredient present in the previously sterile-filtered solution.

[0453] For topical application, compounds and reagents disclosed herein can be used as liquid or solid applications. However, it is generally desired to be topically applied to the skin as a composition in combination with a dermatologically acceptable carrier (which can be solid or liquid). Compounds and reagents disclosed herein and compositions can be topically applied to the skin of a subject to reduce the size of a malignant or benign growth (and can include complete removal) or to treat an infection site. Compounds and reagents disclosed herein can be directly applied to a growth or infection site. Preferably, compounds and reagents are applied to a growth or infection site with preparations such as ointments, creams, lotions, solutions, tinctures, etc.

[0454] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, 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.

[0455] 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 skin of the user.

[0456] 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.

[0457] The dosage range for administering the composition is a dosage range that is large enough to produce the desired effect in which the symptom or disorder is affected. The dosage should not be large enough to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Typically, the dosage will vary with the age, condition, sex, and degree of disease of the patient, and can be determined by those skilled in the art. The dosage can be adjusted by an individual physician in any antagonistic situation. The dosage can vary, and one or more doses can be administered daily, for one day or several days.

[0458] Also disclosed is a pharmaceutical composition comprising a compound disclosed herein in combination with a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a certain amount of a compound suitable for oral, topical or parenteral administration constitutes a preferred aspect. 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 causes no more than an acceptable level of side effects or morbidity. Those skilled in the art will recognize that the 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 treatment ratio, and the severity and stage of the pathological state.

[0459] Also disclosed is a kit comprising a compound disclosed herein in one or more containers. The disclosed kit may optionally include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other components, auxiliary agents or adjuvants 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 compound or composition of the kit. The container of the kit can be any suitable material, such as glass, plastic, metal, etc., and is any suitable size, shape or configuration. In one embodiment, the compound disclosed herein and / or the reagent are provided as a solid (e.g., tablet, pill or powder form) in the kit. In another embodiment, the compound disclosed herein and / or the reagent are provided as a liquid or solution in the kit. In one embodiment, the kit contains an ampoule or syringe containing a compound disclosed herein and / or the reagent in liquid or solution form.

[0460] A number of 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 appended claims. Example

[0461] The following examples are set forth to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalent variants and variations that are obvious to those skilled in the art.

[0462] Efforts have been made to ensure the accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be considered. Unless otherwise indicated, parts are by weight, temperatures are ℃ or ambient temperature, and pressures are at or near atmospheric pressure. There are multiple variations and combinations of reaction conditions (e.g., component concentrations, temperature, pressure, and other reaction ranges and conditions) that can be used to optimize the product purity and yield obtained from the method. Only reasonable and routine experiments are required to optimize such process conditions.

[0463] Example 1

[0464] The cyclic heptapeptide ring (FΦRRRRQ) (cFΦR4, where Φ is L-2-naphthylalanine) was found to be efficiently internalized by mammalian cells. In this study, the mechanism of its internalization was investigated by interfering with various endocytic events through the introduction of pharmacological agents and genetic mutations. The results showed that cFΦR4 can directly bind to membrane phospholipids, can be internalized into human cancer cells by endocytosis, and can escape from early endosomes to the cytoplasm. Its cargo capacity was detected with various molecules, including small molecule dyes, linear and cyclic peptides of various charge states, and proteins. Depending on the nature of the cargo, they can be delivered through the intracyclic (insertion of cargo into the cFΦR4 ring), the exocyclic (cargo attached to the Gln side chain), or the bicyclic route (fusion of cFΦR4 and the cyclic cargo ring). The overall delivery efficiency of cFΦR4 (i.e., cargo delivery into the cytoplasm and the nucleus) is 4 to 12 times higher than that of non-arginine (R9), HIV Tat-derived peptides (Tat), or membrane-penetrating peptides (Antp). The higher delivery efficiency coupled with superior serum stability, minimal toxicity, and ease of synthesis make cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for studying endosomal escape mechanisms.

[0465] introduction

[0466] The plasma membrane is a major challenge for the development of drugs, especially for biologics such as peptides, proteins and nucleic acids. A potential strategy to disrupt the membrane barrier and deliver biologics into cells is to link them to "cell penetrating peptides (CPPs)". Since the initial observation in the late 1980s that the HIV transactivator of transcription, Tat, was internalized into mammalian cells and activated viral replication (Frankel, AD and Pabo, CO. Cell, 1988, 55, 1189-1193; Green, M and Loewenstein, PM. Cell, 1988, 55, 1179-1188), a large number of CPPs consisting of 6-20 residues have been reported (Langel, Cell-penetrating peptides: methods and protocols, Humana Press, New York, 2011, p xv; Schmidt, N et al. FEBS Lett., 2010, 584, 1806-1813; Futaki, S. Adv. Drug Delivery Rev., 2005, 57, 547-558; Stewart, KM et al. Org. Biomol. Chem., 2008, 6, 2242-2255; Deshayes, S et al. Cell. Mol. Life Sci., 2005, 62, 1839-1849; Goun, EA et al. ChemBioChem, 2005, 7, 1497-1515). PCP has been used to conjugate small molecule drugs (Rothbard, JB et al. Nat. Med., 2000, 6, 1253-1257; Nori, A et al. Bioconjugate Chem., 2003, 14, 44-50), DNA (Hoyer, J and Neundorf, I. Acc. Chem. Res., 2012, 45, 1048-1056; Eguchi, A et al. J. Biol. Chem., 2001, 276, 26204-26210), RNA (Nakase, I et al. Acc. Chem. Res., 2012, 45, 1132-1139; Andaloussi, SE et al. Nucleic Acids Res., 2011, 39, 3972-3987; Jeong, JH et al. Bioconjugate Chem., 2009, 20, 5-14; Muratovska, A and Eccles, MR. FEBS Lett., 2004, 558, 63-68), proteins (Wadia, JS and Dowdy, SF. Adv. Drug Delivery Rev., 2005, 57, 579-596; Pooga, M et al. FASEB J., 2001, 15, 1451-1453; Schwarze, SR et al. Science, 1999, 285, 1569-1572) and nanoparticles (Josephson, L et al. Bioconjugate Chem., 1999, 10, 186-191; Gupta, B et al. Adv. Drug Delivery Rev., 2005, 57, 637-651; Liu, J et al. Biomacromolecules, 2001, 2, 362-8) for delivery into mammalian cells and tissues.Many CPPs show minimal toxicity and immunogenicity at physiologically relevant concentrations (Saar, K et al. Anal. Biochem., 2005, 345, 55-65; Suhorutsenko, J et al. Bioconjugate Chem., 2011, 22, 2255-2262), and incorporation of specific unnatural amino acids (Rueping, M et al. ChemBioChem, 2002, 3, 257-259) and other chemical moieties (Cooley, CB et al. J. Am. Chem. Soc., 2009, 131, 16401-16403; Pham, W et al. Chembiochem, 2004, 5, 1148-1151) has been found to increase stability and cytosolic delivery.

[0467] Despite three decades of research, the fundamental basis of CPP activity remains unclear. Two different and non-mutually exclusive mechanisms have been proposed for CPPs whose primary sequences are characterized by multiple arginine residues. In the first mechanism (direct membrane translocation), arginine guanidine interacts with the phospholipids of the plasma membrane to produce neutral ion pairs that passively diffuse across the membrane (Herce, HD and Garcia, AE. Proc. Natl. Acad. Sci. USA, 2007, 104, 20805-20810; Hirose, H et al. Mol. Ther., 2012, 20, 984-993) or promotes the formation of transient holes that allow CPP to pass through lipid bilayers (Herce, HD et al. Biophys. J., 2009, 97, 1917-1925; Palm-Apergi, C et al. FASEB J., 2009, 23, 214-223). In the second mechanism, CPPs associate with cell surface glycoproteins and membrane phospholipids, are internalized into cells by endocytosis (Richard, JP et al. J. Biol. Chem., 2005, 280, 15300-15306; Ferrari, A et al. Mol. Ther., 2003, 8, 284-294; Fittipaldi, A et al. J. Biol. Chem., 2003, 278, 34141-34149; Kaplan, IM et al. J. Controlled Release, 2005, 102, 247-253; Nakase, I et al. Biochemistry, 2007, 46, 492-501), and are subsequently exported from endosomes into the cytoplasm. In summary, most data show that at low CPP concentrations, cellular uptake occurs primarily through endocytosis, while direct membrane translocation becomes prevalent at concentrations above 10 μM (Duchardt, F et al. Traffic, 2007, 8, 848-866). However, the mechanism of entry and the efficiency of uptake may vary with CPP identity, cargo, cell type and other factors (Mueller, J et al. Bioconjugate Chem., 2008, 19, 2363-2374; Maiolo, JR et al. Biochim. Biophys. Acta., 2005, 1712, 161-172).

[0468] CPPs that enter cells via endocytosis must be expelled from endocytic vesicles in order to reach the cytoplasm. Unfortunately, the endosomal membrane has been shown to be an important barrier for the cytoplasmic delivery of these CPPs; a negligible portion of the peptides usually escape into the cell interior (El-Sayed, A et al. AAPS J., 2009, 11, 13-22; Varkouhi, AK et al. J. Controlled Release, 2011, 151, 220-228; Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830). For example, even in the presence of the pro-fusogenic hemagglutinin peptide HA2, which has been shown to enhance the release of endosomal cargo, >99% of the Tat-Cre fusion protein remains embedded in the large chromosome 24 hours after initial uptake (Kaplan, IM et al. J. Controlled Release, 2005, 102, 247-253). Recently, two new types of CPPs with improved endosomal escape efficiency have been discovered. Appelbaum et al. showed that folded mini-proteins containing a discrete five-arginine motif can effectively overcome endosomal entrapment and reach the cytoplasm of mammalian cells (Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830). This motif consists of five arginines across three turns of the α-helix, and proteins containing this motif have been identified since early (Rab5 +) endosomes are released into the cell interior. It has also been found that cyclization of certain arginine-rich CPPs enhances their cellular uptake (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431; Lattig-Tunnemann, G et al. Nat. Commun., 2011, 2, 453; Mandal, D et al. Angew. Chem. Int. Ed., 2011, 50, 9633-9637; Zhao, K et al. Soft Matter, 2012, 8, 6430-6433). Amphiphilic small cyclic peptides such as cyclo(FΦRRRRQ) (cFΦR4, where Φ is L-2-naphthylalanine) are internalized by mammalian cells in an energy-dependent manner and enter the cytoplasm and nucleus with an efficiency 2 to 5 times higher than that of non-arginine (R9) (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431). In addition, membrane-impermeable cargoes such as phosphopeptides can be inserted into the cFΦR4 loop, resulting in their delivery to the cytoplasm of target cells. However, the insertion of cargo into the cyclic peptide loop, which is referred to herein as the "endocyclic" delivery method (Figure 1A), is limited to relatively short peptides (≤7 amino acids) because large loops show poor internalization efficiency (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431).

[0469] In order to understand the mechanism of action of cFΦR4 and potentially design more efficient cyclic CPPs, this paper studies the internalization mechanism of cFΦR4 by using artificial membranes and pharmacological agents as well as genetic mutations that interfere with various endocytic events. The data show that cFΦR4 can bind directly to plasma membrane phospholipids and can enter cells through endocytosis. Like mini-proteins that display a five-arginine motif (Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830), cFΦR4 can escape from early endosomes into the cytosol. cFΦR4 is attached to the Gln side chain by the outer ring (cargo; Figure 1B ) or a dual-ring delivery approach (fusion of cFΦR4 and a circular cargo ring; Figure 1C ) was examined for its ability to deliver a variety of cargo molecules, including linear peptides of different charges, cyclic peptides, and large proteins, into the cytoplasm of mammalian cells. cFΦR4 was found to tolerate the size and nature of the cargo and efficiently transport all cargoes tested into the cytoplasm and nucleus of mammalian cells. In addition, cFΦR4 exhibited superior stability against proteolysis relative to linear CPPs, but minimal cytotoxicity. Thus, cFΦR4 provides a practically useful transporter for cytosolic cargo delivery as well as a system for studying the mechanisms of early endosomal release.

[0470] Materials. Reagents used for peptide synthesis were purchased from Advanced ChemTech (Louisville, KY), NovaBiochem (La Jolla, CA), or Anaspec (San Jose, CA). 2,2'-Bipyridyl disulfide, lissamine rhodamine B sulfonyl chloride, fluorescein isothiocyanate (FITC), dexamethasone (Dex), coenzyme A trilithium salt, FITC-labeled dextran (Dex FITC ) and human serum were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture medium, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-EDTA, Hoescht 33342, Alexa488-labeled dextran (dextran Alexa488 ), Dulbecco's phosphate buffered saline (DPBS) (2.67 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 8.06 mM sodium dihydrogen phosphate), and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). PD-10 desalting columns were purchased from GE-Healthcare (Piscataway, NJ). Nuclear staining dye DRAQ5 TM was purchased from ThermoScientific (Rockford, IL), and the cell proliferation kit (MTT) was purchased from Roche (Indianapolis, IN). Anti-phosphotyrosine (pY) antibody (clone 4G10) was purchased from Millipore (Temecula, CA).

[0471] Rink resin LS (100-200 mesh, 0.2 mmol / g) was purchased from Advanced ChemTech. LC-SMCC (succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxyl-[6-amidohexanoate]) was purchased from ThermoScientific (Rockford, IL), while 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1'-rac-glycerol) (sodium salt) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), sphingomyelin (brain, porcine) and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL). Heparan sulfate (HO-03103, batch number HO-10697) was obtained from Celcus Laboratories (Cincinnati, OH).

[0472] Peptide synthesis and labeling. Peptides were synthesized on Rink Resin LS (0.2 mmol / g) using standard Fmoc chemistry. A typical coupling reaction contained 5 equivalents of Fmoc-amino acid, 5 equivalents of 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 10 equivalents of diisopropylethylamine (DIPEA), and was allowed to continue mixing for 75 minutes. After the addition of the last (N-terminal) residue, the allyl group on the C-terminal Glu residue was removed by treatment with Pd(PPh3)4 and phenylsilane (0.1 and 10 equivalents, respectively) in anhydrous DCM (3×15 minutes). The N-terminal Fmoc group was removed by treatment with 20% piperidine in DMF and the peptide was cyclized by treatment with benzotriazol-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate (PyBOP) / HOBt / DIPEA (5, 5 and 10 equiv) in DMF for 3 h. The peptide was deprotected and released from the resin by treatment with 82.5:5:5:5:2.5 (v / v) TFA / thioanisole / water / phenol / ethanedithiol for 2 h. The peptide was triturated with cold ether (3×) and heated at C 18 Purification was performed on column by reverse phase HPLC. The authenticity of each peptide was confirmed by MALDI-TOF mass spectrometry.

[0473] The peptide was labeled with FITC by dissolving the purified peptide (~1 mg) in 300 μL of 1:1:1 (vol / vol) DMSO / DMF / 150 mM sodium bicarbonate (pH 8.5) and mixing with 10 μL of FITC in DMSO (100 mg / mL). 18 The reaction mixture was subjected to reverse phase HPLC on a column to separate the FITC-labeled peptides. Figure 2 ), N ε-4-methoxytrityl-L-lysine was added to the C-terminus. After solid phase peptide synthesis, lysine side chains were selectively deprotected using 1% (v / v) trifluoroacetic acid in CH2Cl2. The resin was incubated overnight with lissamine rhodamine B sulfonyl chloride / DIPEA (5 equivalents each) in DMF. The peptide was completely deprotected, ground with diethyl ether, and purified by HPLC. Dex-labeled peptides were produced by incubating the resin with dexamethasone-21-thiopropionic acid / HBTU / DIPEA (5, 5, and 10 equivalents) in DMF for 3 hours (Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830). The peptide was then deprotected, ground, and purified by HPLC. Bicyclic peptides, phosphocoumarylaminopropionic acid (pCAP) and pCAP-containing peptides (PCP) were synthesized as previously described (Lian, W et al. J. Am. Chem. Soc., 2013, 135, 11990-11995; Mitra, S and Barrios, AM. Bioorg. Med. Chem. Lett., 2005, 15, 5124-5145; Stanford, SM et al. Proc. Natl. Acad. Sci. USA, 2012, 109, 13972-13977). The authenticity of each peptide was confirmed by MALDI-TOF mass spectrometry.

[0474] Preparation of cFΦR4-protein conjugate. PTP1B cDNA was used as a template, and the gene encoding the PTP1 B catalytic domain (amino acids 1-321) was amplified by polymerase chain reaction using 5'-ggaattccatatggagatggaaaaggagttcgagcag-3' and 5'-gggatccgtcgacattgtgtggctccaggattcgtttgg-3' as primers. The resulting DNA fragment was digested with endonucleases Nde I and Sal I and inserted into the prokaryotic vector pET-22b (+) -ybbR (Yin, J et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 15815-15820). The cloning process resulted in the addition of a ybbR tag (VLDSLEFIASKL) to the N-terminus of PTP1B. Expression and purification of ybbR-tagged PTP1B were performed as described previously (Ren, L et al., Biochemistry, 2011, 50, 2339-2356).

[0475] The peptide cFΦR4 containing a C-terminal cysteine ​​(cFΦR4-SH, ~10 μmol; Figure 3) was dissolved in 1 mL of degassed DPBS and mixed with 2,2'-dipyridyl disulfide (5 equivalents) dissolved in acetone (0.5 mL). After 2 hours at room temperature, the reaction product cFΦR4-SS-Py was purified by reverse phase HPLC. The product was incubated with coenzyme A (2 equivalents) in DPBS for 2 hours. The resulting cFΦR4-SS-CoA adduct was purified again by reverse phase HPLC. Green fluorescent protein (GFP) containing an N-terminal ybbR tag (VLDSLEFIASKL) and a C-terminal hexahistidine tag was expressed in Escherichia coli and purified as previously described (Yin, J et al. Proc. Natl. Acad. Sci. USA, 2005, 102, 15815-15820). Next, ybbR-GFP (30 μM), cFΦR4-SS-CoA (30 μM), and phosphopantetheinyl transferase Sfp (0.5 μM) were mixed in 50 mM HEPES (pH 7.4), 10 mM MgCl2 (total volume 1.5 mL) and incubated at 37°C for 15 min. The labeled protein cFΦ R4-SS-GFP ( Figure 3 ) was separated from unreacted cFΦR4-SS-CoA. GFP conjugated to Tat (Tat-SS-GFP) and PTP1B conjugated to cFΦR4 (cFΦR4-PTP1B) were prepared in a similar manner ( Figure 4 ).

[0476] A peptide containing a C-terminal lysine (cFΦR4-Lys, ∼10 μmol; Figure 4 ), deprotected and released from the support, dissolved in degassed DPBS (pH 7.4, 1 mL), and mixed with the bifunctional linker LC-SMCC (5 equivalents) dissolved in DMSO (0.2 mL). After incubation at room temperature for 2 hours, the 18 The reaction product cFΦR4-SMCC ( Figure 4). The product was then mixed with Coenzyme A (2 equivalents) in DPBS and incubated for 2 hours. The resulting cFΦR4-SMCC-CoA adduct was purified again by reverse phase HPLC. Next, ybbR-labeled PTP1B (30 μM), cFΦR4-SMCC-CoA (30 μM) and phosphopantetheinyl transferase Sfp (0.5 μM) were mixed in 50 mM HEPES (pH 7.4), 10 mM MgCl2 (total volume 1.5 mL) and incubated at 37°C for 15 minutes. The labeled proteins (cFΦR4-PTP1B; Figure 4 ) was separated from unreacted cFΦR4-SMCC-CoA.

[0477] Cell culture and transfection. HEK293, HeLa, MCF-7, NIH 3T3, and A549 cells were maintained in medium consisting of DMEM, 10% FBS, and 1% penicillin / streptomycin. Jurkat, H1650, and H1299 cells were grown in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were cultured in a humidified incubator at 37°C, 5% CO2. For HeLa cell transfection, cells were seeded in 96-well plates at a density of 10,000 cells / well. After attachment, the cells were incubated with proteins encoding Rab5-green fluorescent protein fusion (Rab5-GFP), Rab7-GFP (Addgene plasmid #28047), glucocorticoid receptor (C638G)-GFP fusion (GR-GFP) (Holub, JM et al. Biochemistry, 2013, 50, 9036-6046), DsRed-Rab5 WT (Addgene plasmid #13050), or DsRed-Rab5 Q79L Cells were transfected using Lipofectamine 2000 (Addgene plasmid #29688) following the manufacturer's protocol.

[0478] Preparation of small unilamellar vesicles (SUV). SUVs were prepared by modifying a previously reported method (Magzoub, M et al. Biochim. Biophys. Acta, 2002, 1563, 53-63). The appropriate lipid mixture was dissolved in chloroform in a test tube. The lipid mixture was gently dried by blowing argon over the solution and kept in a desiccator overnight. The dried lipids were rehydrated in DPBS to a final total lipid concentration of 10 mM. The suspension was vigorously mixed by vortexing and ultrasonic treatment on ice until it became clear. A typical preparation produces a homogeneous solution of vesicles containing an average diameter of ~80 nm and a polydispersity (PdI) index <0.15 as determined by dynamic light scattering measurements using a ZetaSizer Nano Series (Malvern, Brookhaven, CT). The SUV solution was stored at 4°C and used for FP experiments on the same day.

[0479] Fluorescence polarization. A typical experiment was performed by incubating 100 nM FITC-labeled peptide with different concentrations of heparan sulfate (0-5,000 nM) in DPBS for 2 h at room temperature. FP values ​​were measured on a Molecular Devices Spectramax M5 spectrofluorimeter with excitation and emission wavelengths of 485 nm and 525 nm, respectively. EC was determined by plotting FP values ​​as a function of heparan sulfate concentration and fitting to a four-parameter logistic curve using GraphPad PRISM ver.6 software. 50 .

[0480] To obtain EC of CPPs with lipid membrane 50 FP values ​​were measured, plotted and analyzed similarly using 100 nM FITC-labeled peptides and increasing concentrations of SUV solutions (0-10 mM) in DPBS.

[0481] Image analysis.Use imageJ to modify the original image consistently.Use Just Another ColocalizationPlugin (JACoP) (Bolte, S and Cordelieres, FP.J.Microsc., 2006,224,213-232) to obtain Pearson correlation coefficient (R) from the endosomal region.For GR-GFP translocation determination, separate GFP and Hoescht images are loaded into a customized CellProfiler pipeline and colored in gray (Carpenter, AE et al. Genome Biol., 2006,7, R 100).The nucleus is distinguished from the Hoescht image via Otsu automatic three-class thresholding, and the pixels of the intermediate intensity category are assigned to the background.The aggregated objects are identified using Laplace Gaussian modeling and separated by shape.The nuclear region is defined as the diameter of the Hoescht object reduced by 1 μm, and the cytoplasmic ring region is defined as the region between the nuclear diameter and the nuclear diameter expanded by 2 μm. The translocation ratio was defined as the mean GFP signal inside the nuclear region divided by the mean GFP signal within the cytosolic region measured for each cell, and 30–70 cells from 15–30 images were captured for each condition tested.

[0482] Confocal microscopy. To examine the rhodamine-labeled cyclic peptide (cFΦR4 Rho ) and Rab5 + or Rab7 + To investigate the colocalization between endosomes, HeLa cells transfected with Rab5-GFP or Rab7-GFP were plated one day before the experiment (200 μL, 10 4 On the day of the experiment, 1 μM cFΦR4 was used in DMEM medium supplemented with 300 nM Hoescht 33342. Rho HeLa cells were treated for 30 min, then washed with HKR buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2) and imaged using a PerkinElmer LiveView spinning disk confocal microscope.

[0483] For GR translocation assays, HeLa cells transfected with GR-GFP were plated as described above (Holub, JM et al. Biochemistry, 2013, 50, 9036-6046). Cells were treated with DMEM medium containing 1 μM Dex or Dex-peptide conjugate and 300nM Hoescht33342 for 30 minutes and imaged using a Zeiss Axiovert 200M epifluorescence microscope equipped with a Ziess Axiocam mRM camera and an EXFO-Excite series 120Hg arc lamp. To study the effects of endocytosis inhibitors, transfected HeLa cells were pretreated with clarified DMEM containing inhibitors for 30 minutes before incubation with Dex or Dex-peptide conjugates. To test whether Rab5 activity is required for endosomal escape, GR-GFP and DsRed-Rab5 WT or DsRed-Rab5 Q79L HeLa cells were transfected, then treated with Dex or Dex-peptide conjugates, and imaged as described above (Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830).

[0484] To study the internalization of rhodamine-labeled peptides, 5 × 10 4 HEK293 cells were plated in 35 mm glass-bottom microwell dishes (MatTek). On the day of the experiment, the cells were incubated with peptide solution (5 μM) and 0.5 mg / mL dextran at 37°C. FITC The cells were incubated for 2 hours. The cells were gently washed twice with DPBS and imaged on a Visitech Infinity 3Hawk 2D-array live cell imaging confocal microscope. To detect the internalization of pCAP-containing peptides, HEK293 cells were similarly plated and incubated with peptide solutions (5 μM) at 37°C for 60 minutes. After removing the culture medium, the cells were gently washed twice with DPBS containing sodium pervanadate (1 mM) and incubated in DPBS containing 5 μM nuclear staining dye DRAQ5 for 10 minutes. The resulting cells were washed twice with DPBS and imaged on a spinning disk confocal microscope (UltraView Vox CSUX1 system). To monitor GFP internalization, 5×10 4 HEK293 cells were seeded in 35 mm glass-bottom microwell dishes and cultured overnight. Cells were treated with cFΦR4-SS-GFP (1 μM) for 2 hours at 37°C. After removing the culture medium, cells were incubated in DPBS containing 5 μM DRAQ5 for 10 minutes. Cells were washed twice with DPBS and imaged on a Visitech Infinity 3Hawk 2D-Array live cell imaging confocal microscope.

[0485] Flow cytometry. To quantify the delivery efficiency of pCAP-containing peptides, HeLa cells were plated in six-well plates (5 × 10 5 Cells were cultured in PBS (10 μM pCAP peptide) for 24 hours. On the day of the experiment, cells were incubated with 10 μM pCAP peptide in clarified DMEM with 1% FBS at 37°C for 2 hours. Cells were washed with DPBS containing 1 mM sodium pervanadate, detached from the plate with 0.25% trypsin, suspended in DPBS containing 1% bovine serum albumin, and analyzed on a BD FACS Aria flow cytometer at 355 nm excitation. Data were analyzed using Flowjo software (Tree Star).

[0486] To evaluate the effect of cFΦR4 on endocytosis, HeLa cells were seeded in 6-well plates (5 × 10 5 After attachment, cells were cultured under standard cell culture conditions with no supplements, 1 μM cFΦR4 peptide, 100 μM dextran Alexa488 (Life Technologies, D-22910) or 1 μM cyclic peptide and 100 μM dextran Alexa488 The cells were treated with clarified DMEM for 30 minutes. The cells were washed twice with DPBS, removed from the plate with 0.25% trypsin, diluted into clarified DMEM containing 10% FBS, pelleted at 300 g for 5 minutes, washed once with DPBS, and resuspended in 200 μL DPBS. Whole cell dextran uptake was analyzed on a BD Accuri C6 flow cytometer using the manufacturer FL1 laser and filter set.

[0487] Immunoblotting. NIH3T3 cells were cultured in complete growth medium to reach 80% confluence. Cells were starved in serum-free medium for 3 hours and treated with different concentrations of cFΦ R4-PTP1B or unlabeled PTP1B for 2 hours, followed by incubation in medium supplemented with 1 mM sodium pervanadate for 30 minutes. The solution was removed and the cells were washed twice with cold DPBS. Cells were detached and lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 10 mM sodium pyrophosphate, 5 mM iodoacetic acid, 10 mM NaF, 1 mM EDTA, 2 mM sodium pervanadate, 0.1 mg / mL phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 0.1 mg / mL trypsin inhibitor. After incubation on ice for 30 minutes, the cell lysate was centrifuged at 15,000 rpm in a microcentrifuge for 25 minutes. Total cellular proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes and immunoblotted using anti-pY antibody 4G10.

[0488] Serum stability test. Stability testing was performed by modifying a previously reported procedure (Nguyen, LT et al. PLoS One, 2010, 5, e12684). Diluted human serum (25%) was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected. The peptide stock solution was diluted into the supernatant to a final concentration of 5 μM for cFΦR4 and Antp, and 50 μM for peptide R9 and Tat, and incubated at 37°C. At various time points (0-6 hours), a 200-μL aliquot was removed and mixed with 50 μL of 15% trichloroacetic acid and incubated overnight at 4°C. The final mixture was centrifuged at 15,000 rpm for 10 minutes in a microcentrifuge and precipitated by a 4% 4% 4% 4% 2 ... 18 The supernatant was analyzed by reverse phase HPLC on a 50% column (Waters). The amount (%) of remaining peptide was determined by integrating the area under the peptide peak (monitored at 214 nm) and compared with that of a control reaction (without serum).

[0489] Cytotoxicity assay. MTT assay was performed to evaluate the cytotoxicity of cyclic peptides to several mammalian cell lines (Mosmann, TJ Immunol. Methods, 1983, 65, 55-63). 100 μL of MCF-7, HEK293, H1299, H1650, A549 (1×10 5 Cells (cells / mL) were placed in each well of a 96-well culture plate and grown overnight. Different concentrations of peptide (5 or 50 μM) were added to each well and the cells were incubated at 37°C with 5% CO2 for 24 to 72 hours. 10 μL of MTT stock solution was added to each well. 10 μL of solution was added to the growth medium (without cells) as a negative control. The plate was incubated at 37°C for 4 hours. 100 μL of SDS-HCl solubilization buffer was then added to each well and the resulting solution was mixed thoroughly. The plate was incubated at 37°C for another 4 hours. The absorbance of the formazan product was measured at 570 nm using a Molecular Devices Spectramax M5 plate reader. Each experiment was performed in triplicate, and cells without any peptide were treated as controls.

[0490] cFΦR4 binds to membrane phospholipids. It was previously observed that 1 μM FITC-labeled cyclic peptide cFΦR4 was incubated with vesicles containing negatively charged phospholipids (90% phosphatidylcholine (PC) and 10% phosphatidylglycerol (PG)) FITCResulting in peptide fluorescence quenching, which is consistent with direct binding of cFΦR4 to phospholipids (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431). To test the potential role of membrane binding during endocytic uptake of CPPs, SUVs that mimic the outer membrane of mammalian cells (45% PC, 20% phosphatidylethanolamine, 20% sphingomyelin, and 15% cholesterol) were prepared and tested for binding to FITC-labeled cFΦR4, R9, and Tat (all at 100 nM) by fluorescence polarization (FP) assay. cFΦR4 was expressed as EC 50 Value (combined with cFΦR4 FITC The lipid concentration of half of the total lipid concentration) was 2.1 ± 0.1 mM bound to neutral SUV ( Figure 5A ). R9 showed much weaker binding to artificial membranes (EC 50 >10 mM), while Tat did not bind at all. Next, the CPPs were tested for binding to heparan sulfate, which has been previously proposed as the primary binding target of cationic CPPs (Nakase, I et al. Biochemistry, 2007, 46, 492-501; Rusnati, M et al. J. Biol. Chem., 1999, 274, 28198-28205; Tyagi, M et al. J. Biol. Chem., 2001, 276, 3254-3261; Ziegler, A and Seelig, J. Biophys. J., 2004, 86, 254-263; Goncalves, E et al. Biochemistry, 2005, 44, 2692-2702; Ziegler, A. Adv. Drug Delivery Rev., 2008, 60, 580-597). Both R9 and Tat bind to heparan sulfate with high affinity, EC 50 The values ​​were 144nM and 304nM (Fig. 5B) respectively. Under the same conditions, cFΦR4 showed that it could not detect the combination with heparan sulfate. These results are consistent with previous observations, that is, non-amphipathic cationic CPP (such as Tat and R9) tightly binds to cell surface proteoglycans (such as heparan sulfate), but only weakly binds to membrane lipids (Ziegler, A.Adv.Drug Delivery Rev., 2008, 60, 580-597). The lack of positive charge number of cFΦR4 may be due to its lack of strong electrostatic interaction with heparan sulfate. On the other hand, the amphipathic nature and more rigid ring structure of cFΦR4 should help it to bind to neutral lipid membranes. These data, together with the above-mentioned inhibition mode by various endocytosis inhibitors, show that cFΦR4 can be directly bound to plasma membrane phospholipids and can be internalized by all endocytosis mechanisms in a piggyback manner.

[0491] Intracellular delivery of peptide-based cargoes. Since the intracyclic delivery of cFΦR4 is limited to cargoes of heptapeptides or smaller (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431), in this study, the ability of cFΦR4 to deliver cargoes of different sizes and physicochemical properties attached to the Gln side chain (Figure 1B, extracyclic delivery) was tested. First, positively charged (RRRRR), neutral (AAAAA), hydrophobic (FFFF), and negatively charged peptides [DE(pCAP)LI] were covalently attached to cFΦR4. The first three peptides were labeled with rhodamine B at the C-terminal lysine side chain (Figure 2), and their internalization into HEK293 cells was detected by live cell confocal microscopy. 5 μM peptide cFΦR4-A5 ( Fig. 6A ) or cFΦR4-R5( Figure 6B ) Incubating cells for 2 hours showed evidence of both punctate fluorescence and diffuse fluorescence, the latter being almost uniformly distributed throughout the cell. In contrast, the fluid-phase endocytosis marker dextran FITC Predominantly punctate fluorescence was shown, indicating endosomal localization. Diffuse rhodamine fluorescence indicated that a portion of the peptide reached the cytosol and nucleus of the cell. Cells were incubated with cFΦR4 (1 μM) and dextran Alexa488 Co-incubation with 1% cytosolic markers increased internalization by 15% ( Figure 7 ), indicating that cFΦR4 can activate endocytosis in cultured cells. cFΦR4-F4 was not tested due to its poor water solubility.

[0492] Designed peptide cFΦR4-DE(pCAP)LI(cFΦR4-PCP; Figure 2) to test the ability of cFΦR4 to deliver negatively charged cargoes and to compare the cytoplasmic delivery efficiency of cFΦR4 with other widely used CPPs such as R9, Tat, and transmembrane peptide (Antp). Therefore, unlabeled PCP [Ac-DE(pCap)LI-NH2] and PCP conjugated with R9 (R9-PCP), PCP conjugated with Tat (Tat-PCP), or PCP conjugated with Antp (Antp-PCP) were also prepared. Note that cFΦR4-PCP carries a net charge of zero at physiological pH. pCAP is non-fluorescent, but after entering the cell interior, it should be rapidly dephosphorylated by endogenous protein tyrosine phosphatases (PTPs) to produce a fluorescent product, coumaroylaminopropionic acid (CAP, excitation 355 nm; emission 450 nm) (Mitra, S and Barrios, AM. Bioorg. Med. Chem. Lett., 2005, 15, 5124-5145; Stanford, SM et al. Proc. Natl. Acad. Sci. USA, 2012, 109, 13972-13977). When assayed against the PTP group in vitro, all four CPP-PCP conjugates were effectively dephosphorylated (Table 8). This assay detects only CPP-cargoes in the cytoplasm and nucleus, where the catalytic domains of all known mammalian PTPs are located (Alonso, A et al. Cell, 2004, 117, 699-711). Furthermore, CAP is fluorescent only in its deprotonated state (pKa = 7.8); even if some dephosphorylation occurs within endosomes (pH 6.5-4.5) or lysosomes (pH 4.5), its contribution to the overall fluorescence is minimal ( Figure 8 Treatment of HEK293 cells with 5 μM cFΦR4-PCP for 60 min resulted in diffuse blue fluorescence throughout the cells, indicating that cFΦR4-PCP reached the interior of the cells, whereas unlabeled PCP could not enter the cells under the same conditions ( Fig. 9A When HEK293 cells were pretreated with the PTP inhibitor sodium pervanadate for 1 h and then incubated with cFΦR4-PCP (5 μM), CAP fluorescence in the cells was reduced to background levels. HEK293 cells treated with R9-PCP, Antp-PCP, or Tat-PCP under the same conditions showed weak fluorescence, which is consistent with the poor ability of these peptides to access the cell interior ( Fig. 9A To quantify the relative intracellular PCP delivery efficiency, HeLa cells were treated with each peptide and analyzed by fluorescence activated cell sorting ( Fig. 9BcFΦR4-PCP was most efficiently internalized by HeLa cells with a mean fluorescence intensity (MFI) of 3510 arbitrary units (AU), while R9-PCP, Antp-PCP, Tat-PCP, and unlabeled PCP produced MFI values ​​of 960, 400, 290, and 30 AU, respectively ( Fig. 9C ). In addition, when cells were treated with cFΦR4-PCP in the presence of sodium pervanadate, the amount of CAP fluorescence decreased to close to the background level (70 AU). Therefore, cFΦR4 is able to deliver peptide cargoes with different physicochemical properties into the cytoplasm with an efficiency 3.7-12 times higher than that of R9, Antp, and Tat.

[0493] Table 8. Kinetic activity (k) of recombinant PTPs against pCAP-containing peptides cat / K M , M -1 s -1 ) a

[0494]

[0495] a k was measured as previously described (Ren, L et al. Biochemistry, 2011, 50, 2339-2356) cat / K M .

[0496] Intracellular delivery of cyclic peptides. In recent years, there has been great interest in cyclic peptides as therapeutic agents and biomedical research tools (Driggers, EM et al. Nat. Rev. Drug Discov., 2008, 7, 608-624; Marsault, E and Peterson, ML. J. Med. Chem., 2011, 54, 1961-2004). For example, cyclic peptides are effective for inhibiting protein-protein interactions (Lian, W et al. J. Am. Chem. Soc., 2013, 135, 11990-11995; Liu, T et al. ACS Comb. Sci., 2011, 13, 537-546; Dewan, V et al. ACS Chem. Biol., 2012, 7, 761-769; Wu, X et al. Med. Chem. Commun., 2013, 4, 378-382), which are challenging targets for conventional small molecules. The main obstacle to the development of cyclic peptide therapeutics is that they are generally impermeable to cell membranes (Kwon, YU and Kodadek, T. Chem. Biol., 2007, 14, 671-677; Rezai, T et al. J. Am. Chem. Soc., 2006, 128, 2510-2511; Chatterjee, J et al. Acc. Chem. Res., 2008, 41, 1331-1342). Attempts to deliver cyclic peptides via cFΦR4 via an intracyclic approach have only achieved limited success; increasing the size of the cargo by 1 to 7 residues resulted in progressively poorer cellular uptake, probably because the larger, more flexible ring binds poorly to the cell membrane (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431). To overcome this limitation, bicyclic peptide systems have been investigated, in which one ring contains a CPP motif (e.g., FΦR4) and the other ring consists of a peptide sequence specific for the desired target ( Figure 1C). The bicyclic system should in principle be able to accommodate cargo of any size, because the cargo does not change the structure of the CPP ring and should have a smaller effect on its delivery efficiency. The additional rigidity of the bicyclic structure should also improve its metabolic stability and target binding affinity and specificity. Bicyclic peptides are easily synthesized by forming three amide bonds (i.e., the side chain of N-terminal amine, C-terminal diaminopropionic acid (Dap) and the side chain of lysine (or ornithine, Dap) embedded between CPP and target binding motifs) between the three amino groups on the trimesoyl scaffold and the corresponding linear peptide (Lian, W et al. J. Am. Chem. Soc., 2013, 135, 11990-11995). In order to test the effectiveness of this method, FΦR4 was selected as the CPP part in the C-terminal ring, and peptides of different lengths and charges (AAAAA, AAAAAAA, RARAR or DADAD) were selected as cargo (Table 8, compounds 13-16). For comparison, two single-ring peptides containing FΦR4 as transporter and peptides A5 and A7 as cargo were also prepared (Table 8, compounds 17 and 18). All peptides were labeled with rhodamine B at the C-terminal lysine side chain ( Figure 2 ), and their internalization into HEK293 cells was detected by live cell confocal microscopy. Treatment of cells with 5 μM peptides for 2 hours resulted in efficient internalization of all six peptides ( FIG. 10 ), but FACS analysis showed that the bicyclic (FΦR4-A5) Rho The uptake of the peptides was about 3 times more efficient than that of the corresponding single-ring peptide (compound 17). The intracellular distribution of the internalized peptides was quite different between the bicyclic peptides and the single-ring peptides. While the four bicyclic peptides showed evidence of their presence in the cytoplasm / nucleus (as indicated by diffuse rhodamine fluorescence) and endosomes (as indicated by fluorescent spots), the single-ring peptides showed mainly association with the endocytic marker dextran. FITC Overlapping punctate fluorescence. In all cases, the endocytic marker showed only punctate fluorescence, indicating that endosomes were intact in cells treated with the peptides. These results suggest that the increased structural rigidity of the bicyclic peptides facilitates initial uptake and endosomal release via endocytosis, likely due to their improved binding to plasma and endosomal membranes. The bicyclic system may provide a general strategy for intracellular delivery of cyclic and bicyclic peptides.

[0497] Intracellular delivery of protein cargo. To test whether cFΦR4 is able to transport full-length proteins into mammalian cells, GFP was linked to the N-terminus of cFΦR4 via a disulfide bond ( Fig.11A and Figure 3). GFP was chosen because of its intrinsic fluorescence. The disulfide exchange reaction is highly specific, efficient, and reversible; after entering the cytoplasm, the CPP-SS-protein conjugate can be rapidly reduced to release the native protein. Although cFΦR4 can be directly linked to native or engineered surface cysteine ​​residues on the cargo protein, a GFP variant containing a 12 amino acid ybbR tag at its N-terminus was used, and the cFΦR4 was enzymatically linked to the ybbR tag using the phosphopantetheine transferase Sfp (Yin, J et al. Proc. Natl. Acad. Sci. USA, 2005, 102, 15815-15820). This makes it possible to link a single cFΦR4 unit to GFP in a site-specific manner. For comparison, a Tat-SS-GFP conjugate was produced in the same manner. Incubation of HEK293 cells in the presence of 1 μM cFΦR4-SS-GFP resulted in the accumulation of intracellular green fluorescence ( Fig. 11B ). The fluorescence signal is diffuse and present throughout the cell volume, but has a higher concentration in the nucleus. Some cells contain small spots of intense green fluorescence (indicated by Fig. 11B ), which may represent endosomally hidden cFΦR4-SS-GFP or aggregated GFP in the cell. Untagged GFP cannot enter the cell, while Tat-SS-GFP enters the cell less efficiently than cFΦR4-SS-GFP ( Fig. 11B ); FACS analysis of HaLa cells treated with 1 μM protein showed that the total intracellular fluorescence of the latter was 5.5 times higher. Fluorescent spots around the cells and the lack of any detectable fluorescence in the nuclear region of Tat-SS-GFP treated cells indicate that Tat-SS-GFP is mainly buried in endosomes, which is consistent with previous reports (Kaplan, IM et al. J. Controlled Release, 2005, 102, 247-253). Therefore, for proteins as cargo, cFΦR4 also has a higher efficiency than Tat in terms of both initial uptake and endosomal escape.

[0498] To demonstrate the generality of protein delivery of cFΦR4, the catalytic domain (amino acids 1-321) of the functional enzyme PTP1B was selected for delivery to the cell interior. To show that non-cleavable linkages are also compatible with the delivery method, cFΦR4 was conjugated to ybbR-tagged PTP1B via a thioether bond (cFΦR4-PTP1B) ( Figure 4In vitro assays using p-nitrophenyl phosphate as a substrate showed that the addition of the cFΦR4 tag did not affect the activity of PTP1B (Table 9). NIH 3T3 cells were incubated for 2 h in the presence of untagged PTP1B or cFΦR4-PTP1B and their global pY protein levels were analyzed by anti-pY western blotting ( Fig. 12A Treatment of cells with cFΦR4-PTP1B, but not untagged PTP1B, resulted in a concentration-dependent decrease in the pY levels of most, but not all, proteins. Total cellular protein levels, as detected by Coomassie blue staining, were unchanged ( Fig. 12B ), indicating that the observed decrease in pY levels is due to dephosphorylation of pY proteins by cFΦR4-PTP1B and / or secondary effects caused by the introduction of cFΦR4-PTP1B (e.g., inactivation of cellular protein tyrosine kinases). Interestingly, different proteins showed different dephosphorylation kinetics. Several proteins in the 150-200 kD range were completely dephosphorylated after the addition of 62 nM cFΦR4-PTP1B, while a protein of approximately 80 kD remained phosphorylated at 500 nM cFΦR4-PTP1B. The change in pY pattern is consistent with the broad substrate specificity of PTP1B (Ren, L et al. Biochemistry, 2011, 50, 2339-2356), and is very similar to that caused by overexpression of PTP1B in the cytosol of mammalian cells (LaMontagne Jr., KR et al. Proc. Natl. Acad. Sci. USA, 1998, 95, 14094-14099). These results indicate that cFΦR4 can deliver PTP1B to the interior of NIH 3T3 cells in a catalytically active form and at a level sufficient to interfere with cell signaling processes. cFΦR4 therefore provides a tool for introducing other functional proteins, particularly proteins that cannot be genetically expressed (e.g., toxic and chemically modified proteins), into mammalian cells to study their cellular functions.

[0499] Table 9. Kinetic activity of PTP1B and cFΦR4-PTP1B against pNPP (k cat / K M , M -1 s -1 ) a

[0500]

[0501] a pNPP = p-nitrophenyl phosphate; k was measured as described above cat / K M(Ren, L et al. Biochemistry, 2011, 50, 2339-2356).

[0502] Stability and cytotoxicity of cFΦR4. The relative stability of cFΦR4, R9, Tat and Antp (Table 8, compounds 19-22) against proteolytic degradation was determined by incubating the CPPs in 25% human serum at 37°C and following the disappearance of the full-length peptides by reverse phase HPLC. The cationic tryptophan-containing peptide Antp was the least stable of the four CPPs; it degraded with a half-life of <20 minutes and was completely digested after 2 hours ( Fig.13A ). R9 and Tat are slightly more stable than Antp, with a half-life of about 30 minutes. In contrast, cFΦR4 is very stable to serum proteases. Less than 10% degradation after 6 hours of incubation; >70% of cFΦR4 remained intact after 24 hours of incubation in serum. Many other studies have also demonstrated that cyclization of peptides improves their proteolytic stability (Nguyen, LT et al. PLoS One, 2010, 5, e12684). The potential cytotoxicity of cFΦR4 was evaluated by MTT assay using five different human cell lines (HEK293, MCF-7, A549, H1650, and H1299). There was no significant growth inhibition for any cell line after incubation with up to 50 μM cFΦR4 for 24 or 48 hours ( Fig. 13B After 72 hours, slight growth inhibition (up to 20%) was observed at 50 μM ( FIG. 14 ). Thus, cFΦR4 is relatively nontoxic to mammalian cells.

[0503] In this study, it was demonstrated that cFΦR4 can effectively deliver small molecules, peptides, and protein cargoes exocyclically into the cytoplasm and nucleus of mammalian cells. By using a peptide containing pCAP as a cargo / reporter, it was shown that cFΦR4 can be 3.7-12 times more efficient than R9, Tat, and Antp for cytoplasmic cargo delivery, making cFΦR4 one of the most active CPPs known to date. Although there are modifications of polybasic CPPs, such as the addition of hydrophobic acyl groups, which have been previously reported to increase cellular uptake by a similar magnitude (Pham, W et al. Chembiochem, 2004, 5, 1148-1151), these previous studies have not yet determined whether the enhanced uptake translates into a similar increase in cytoplasmic CPP concentrations. The pCAP-based reporter system described herein may provide a simple, robust method to quantitatively assess the cytoplasmic delivery efficiency of other CPPs. Several lines of evidence suggest that cFΦR4 can enter cells through a variety of endocytic mechanisms, including its inability to enter cells at 4°C or in the presence of sodium azide, and that cFΦ R4 Rho Fluorescent spots and fluid phase endocytosis marker dextranFITC The partial overlap between Rho Colocalization with endosomal proteins Rab5 and Rab7, and decreased cFΦR4 after administration of endocytosis inhibitors Dex In addition to cFΦR4 and Rab5 + In addition to the strong colocalization observed between endosomes, the minimal effects of the PI3K inhibitor wortmannin and the Rab5 Q79L mutation on the cytoplasmic delivery of cFΦR4 suggest that cFΦR4 can escape from early endosomes ( Fig.15 ). In contrast, Tat has been shown to enter cells by endocytosis and be released from late endosomes, whereas R9 escapes to endosomes before Rab7 recruitment (Appelbaum, JS et al. Chem. Biol., 2012, 19, 819-830). Early endosomal release can provide advantages, particularly for peptide and protein cargoes, because it can minimize cargo degradation by late endosomal and lysosomal proteases and denaturation caused by acidification during endosomal maturation. In fact, both GFP and PTP1B delivered to the cytoplasm by cFΦR4 are in their folded active forms, as evidenced by green fluorescence and the ability to dephosphorylate intracellular pY proteins, respectively. In addition, due to its more rigid structure, cFΦR4 can be more stable than linear peptides against proteolytic degradation, and due to its smaller size, cFΦR4 can be less expensive to synthesize and may be less likely to interfere with cargo function. These properties can make cFΦR4 a useful transporter for cytosolic delivery of small molecules to protein cargoes. Direct protein delivery can provide a useful research tool, for example, for studying the cellular function of proteins, as it can provide improved temporal control over DNA transfection and subsequent gene expression, and can enable the delivery of chemically modified proteins and proteins whose overexpression can cause toxicity. The ability of cFΦR4 to escape from early endosomes and its simple structure can also provide an excellent system for elucidating endosomal escape mechanisms and factors that influence escape efficiency.

[0504] Example 2

[0505] Cyclization of peptide ligands can effectively improve their stability to proteolytic degradation and, in some cases, their cell permeability. However, this strategy is incompatible with proteins that recognize peptide ligands in extended conformations (e.g., β-strands and α-helices). In this work, a general strategy for intracellular delivery of linear peptide ligands was developed by fusing them to an amphipathic sequence motif (e.g., RRRRΦF, where Φ is L-naphthylalanine) and cyclizing the resulting conjugate via a disulfide bond. Cyclized peptides can have enhanced proteolytic stability and membrane permeability; upon entering the cytoplasm / nucleus of the cell, disulfide bonds can be cleaved to release linear bioactive peptides by reducing the intracellular environment. This strategy is suitable for the generation of cell-permeable peptides as caspase substrates and inhibitors against the CAL PDZ domain for potential treatment of cystic fibrosis.

[0506] The applicability of linear peptides as drugs is generally limited by their susceptibility to proteolytic cleavage and poor membrane permeability. Cyclization of peptides can effectively improve their proteolytic stability (Nguyen, LT et al. PLoS One, 2010, 5, e12684). In addition, it has recently been reported that the cyclization of certain amphipathic peptides (e.g., FΦRRRR, where Φ is L-2-naphthylalanine) can make them cell permeable through an active transport mechanism (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). By incorporating these short sequence motifs into biologically active cyclic peptides, biologically active cyclic peptides can be delivered to the cytoplasm and nucleus of mammalian cells (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). However, in many cases, binding to molecular targets (e.g., PDZ (Doyle, DA et al. Cell 1996, 85, 1067; Morais Cabral, JH et al. Nature 1996, 382, ​​649) and BIR domains (Wu, G et al. Nature 2000, 408, 1008)) may require the peptidyl ligand to exist in its extended conformation (e.g., α-helix and β-strand), and cyclization may interfere with target binding. In this article, a potential general strategy for delivering linear peptide ligands into mammalian cells via reversible disulfide bond-mediated cyclization was studied. When present in an oxidative extracellular environment, the peptide can exist as a macrocycle, which can have enhanced stability to proteolysis and cellular permeability. After entering the cell (i.e., the cytoplasm and / or nucleus), the disulfide bonds can be reduced by intracellular thiols to produce linear bioactive peptides ( Fig.16 ) (Cascales, L et al. J. Biol Chem. 2011, 286, 36932; Jha, D et al. Bioconj Chem. 2011, 22, 319).

[0507] Materials. Reagents used for peptide synthesis were purchased from Advanced ChemTech (Louisville, KY), NovaBiochem (La Jolla, CA), or Anaspec (San Jose, CA). Rink resin LS (100-200 mesh, 0.2 mmol / g) was purchased from Advanced Chem Tech. Dextran Rho , trypsin and α-chymotrypsin were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture medium, fetal bovine serum, penicillin-streptomycin, 0.25% trypsin-EDTA and DPBS were purchased from Invitrogen (Carlsbad, CA). Nuclear staining dye DRAQ5 TM Purchased from Thermo Scientific (Rockford, IL). Caspase-3, human recombinant protein was purchased from EMD Chemicals (San Diego, CA).

[0508] Peptide Synthesis. Most peptides were synthesized on Rink Resin LS (0.2 mmol / g) using standard Fmoc chemistry. A typical coupling reaction contained 5 equivalents of Fmoc-amino acid, 5 equivalents of 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 10 equivalents of diisopropylethylamine (DIPEA), and was allowed to continue mixing for 75 minutes. The peptides were deprotected and released from the resin by treatment with 92.5:2.5:2.5:2.5 (v / v) trifluoroacetic acid (TFA) / water / phenol / triisopropylsilane (TIPS) for 2 hours. The peptides were triturated with cold ether (3×) and precipitated by a 50% HPLC-MS / MS setup equipped with C 18 The peptides were labeled with fluorescein isothiocyanate (FITC) by dissolving the purified peptides (about 1 mg each) in 300 μL of 1:1:1 DMSO / DMF / 150 mM sodium bicarbonate (pH 8.5) and mixing with 10 μL of FITC in DMSO (100 mg / mL). After 20 minutes at room temperature, the peptides were incubated at 4 °C for 24 hours. 18 The reaction mixture was subjected to reverse phase HPLC on a column to separate the FITC-labeled peptide.

[0509] To generate disulfide-mediated cyclic peptides, after removing the N-terminal Fmoc protecting group by treatment with 20% (v / v) piperidine in DMF, 3,3'-dithiodipropionic acid (10 equivalents) was coupled to the N-terminus using 10 equivalents of N,N'-diisopropylcarbodiimide (DIC) and 0.1 equivalents of 4-(dimethylamino)pyridine (DMAP) in anhydrous DCM for 2 hours. The resin was then incubated in 20% β-mercaptoethanol in DMF for 2 hours, twice, to expose the free thiol. The ground crude linear peptide was incubated overnight in 5% DMSO in PBS buffer at pH 7.4 (Tam, JP et al. J. Am. Chem. Soc. 1991, 113, 6657), then ground and HPLC purified as described above (Tam, JP et al. J. Am. Chem. Soc. 1991, 113, 6657).

[0510] To produce thioether-mediated cyclic peptides, after removing the N-terminal Fmoc protecting group by treating with 20% (v / v) piperidine in DMF, 4-bromobutyric acid (10 equivalents) was coupled to the N-terminus using 10 equivalents of DIC and 0.1 equivalents of DMAP in anhydrous DCM for 2 hours. 1% trifluoroacetic acid (TFA) in DCM was used to selectively remove the 4-methoxytrityl (Mmt) protecting group on the side chain of L-cysteine. Thioether formation was performed by incubating the resin in 1% DIPEA in DMF overnight under nitrogen protection. The cyclized peptide was then ground and purified as described above (Roberts, KD et al. Tetrahedron Lett. 1998, 39, 8357).

[0511] Fluorescent caspase substrates were synthesized using Fmoc-Asp(Wang-resin)-AMC (AMC=7-amino-4-methylcoumarin) (NovaBiochem) as solid support. Standard Fmoc chemistry was used to synthesize peptides on the solid phase. The peptides were released from the resin by treatment with 95:2.5:2.5 (v / v) TFA / phenol / water for 2 hours (Maly, DJ et al. J. Org. Chem. 2002, 67, 910).

[0512] Cell culture. HeLa cells were maintained in a medium consisting of DMEM, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Jurkat cells were maintained in a medium consisting of RPMI-1640, 10% FBS and 1% penicillin / streptomycin. Bronchial epithelial CFBE cell lines homozygous for the ΔF508-CFTR mutation were maintained in DMEM containing L-glutamine supplemented with 10% FBS and 1% penicillin / streptomycin. Human fibronectin (1 mg / ml), bovine collagen I (3 mg / ml) and bovine serum albumin (1 mg / ml) were used to coat tissue culture plates. Cells were cultured in a humidified incubator at 37°C, 5% CO2.

[0513] Confocal microscopy. To detect peptide internalization, 1 mL of HeLa cell suspension (5 × 10 4 Cells were seeded in 35 mm glass-bottom microwell dishes (MatTek) and cultured overnight. The cells were gently washed twice with DPBS and incubated with FITC-labeled peptides (5 μM) and dextran in phenol red-free DMEM containing 1% serum at 37°C in the presence of 5% CO2. Rho (0.5mgmL -1 ) for 1 hour. After removing the culture medium, the cells were gently washed twice with DPBS and incubated with 5 μM DRAQ5 in DPBS for 10 minutes. The cells were washed twice with DPBS again and imaged on a Visitech Infinity 3Hawk 2D-array live cell imaging confocal microscope. Images were captured under the same parameters and adjusted using MetaMorph (Molecular Devices) under the same settings.

[0514] Flow cytometry. In six-well plates (5 × 10 5 HeLa cells were cultured in 4% paraformaldehyde (40% flask cells) for 24 hours. On the day of the experiment, cells were incubated with 5 μM FITC-labeled peptide in clarified DMEM containing 1% FBS at 37°C for 2 hours. Cells were washed with DPBS, detached from the plate with 0.25% trypsin, diluted into clarified DMEM containing 10% FBS, pelleted at 250 g for 5 minutes, washed once with DPBS and resuspended in DPBS containing 1% bovine serum albumin and analyzed on a BD FACS Aria flow cytometer. Data were analyzed using Flowjo software (Tree Star).

[0515] To quantify the delivery efficiency of PCP-conjugated peptides, 5 × 10 5HeLa cells were cultured in 4% PBS (100 μM) for 24 h. On the day of the experiment, cells were incubated with 5 μM pCAP-containing peptides in clarified DMEM containing 1% FBS for 2 h at 37°C. Cells were washed with DPBS containing 1 mM sodium pervanadate, detached from the plate with 0.25% trypsin, suspended in DPBS containing 1% bovine serum albumin, and analyzed on a BD FACS Aria flow cytometer with 355 nm excitation.

[0516] Peptide proteolytic stability assay. Stability test was performed by slightly modifying the previously reported method (Frackenpohl, J et al. Chembiochem 2001, 2, 445). 24 μL of 1.5 mM peptide solution was incubated with 30 μL 50 μM α-chymotrypsin and 30 μL 50 μM trypsin in 200 μL working buffer (50 mM Tris-HCl, pH 8.0, NaCl (100 mM), CaCl2 (10 mM)) at 37°C. 40 μL aliquots were taken out at different time points (0-12 hours) and mixed with 40 μL of 15% trichloroacetic acid and incubated overnight at 4°C. The final mixture was centrifuged at 15,000 rpm for 10 minutes in a microcentrifuge and passed through a centrifuge equipped with C 18 The supernatant was analyzed by reverse phase HPLC on a 50% HPLC column (Waters). The amount (%) of remaining peptide was determined by integrating the area under the peptide peak (monitored at 214 nm) and compared with that of a control reaction (without protease).

[0517] One hour before the experiment, 100 μL of Jurkat cell suspension (5×10 5 Cells / mL) were seeded in 96-well plates. 10 μL of staurosporine stock solution (10 μM) was added to half of the wells to induce apoptosis, while 10 μL of culture medium was added to the other wells. After incubation for 1 hour, caspase-3 fluorescent substrate was added to the cells to a final concentration of 5 μM. The fluorescence of the released coumarin was measured at different time points (0-6 hours) on a Spectramax M5 plate reader at excitation and emission wavelengths of 360 and 440 nm. The increase in fluorescence units (FU) between induced and uninduced cells was plotted against the time of caspase-3 activity measured in real time using various fluorescent substrates in living cells. Three independent experiments were performed, each in triplicate.

[0518] In vitro fluorescence determination. First, 0.5 μL (100U / μL) caspase-3 enzyme was incubated with 90 μL reaction buffer (50mM HEPES, pH 7.4, 100mM NaCl, 10mM DTT) in a 96-well plate for 30 minutes. Fluorescent substrate (10 μL, 100 μM) was mixed into the above solution to start the reaction and the plate was measured on a Spectramax M5 plate reader (Ex=360nm, Em=440nm) (Molecular Devices). The increase in fluorescence units (FU) at 1 minute intervals was associated with the release of Amc due to protease activity. ΔFU / min was calculated from the linear portion of the reaction curve. The reported values ​​are the average of three experiments with the standard deviation shown.

[0519] Fluorescence anisotropy. Complete fluorescence anisotropy (FA) titration experiments were performed by incubating 100 nM fluorophore-labeled peptidyl ligand with different concentrations (0-6 μM) of CAL-PDZ (Cushing, PR et al. Biochemistry 2008, 47, 10084) in FA buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM glutathione, 0.1% (w / v) bovine serum albumin) at room temperature for 2 hours. FA values ​​were measured on a Molecular Devices Spectramax M5 spectrofluorometer with excitation and emission wavelengths of 485 nm and 525 nm, respectively. The equilibrium dissociation constant (K) was determined by plotting the fluorescence anisotropy values ​​as a function of CAL-PDZ concentration. D ). The titration curve was fit to the following equation, which assumes a 1:1 binding stoichiometry

[0520]

[0521] where Y is the anisotropy measured at a given CAL-PDZ concentration x; L is the bicyclic peptide concentration; Q b / Q f is the correction factor for dye-protein interaction; A 最大值 is the maximum anisotropy when all peptides bind to CAP-PDZ, and A 最小值 is the minimum anisotropy when all peptides are free.

[0522] Immunofluorescence staining. Briefly, bronchial epithelial CFBE cells homozygous for the DF508-CFTR mutation were treated with 10 mM Corr-4a in the presence and absence of 50 μM unlabeled peptide 8. After treatment, cells were fixed in cold methanol for 20 minutes. Slides were then incubated in 1% BSA / PBS for 10 minutes, followed by incubation with mouse anti-human monoclonal CFTR antibody (R&D Systems) for 1 hour at 37°C. Thereafter, slides were incubated with Alexa Fluor 500 at 37°C. 488-conjugated anti-mouse IgG2a secondary antibody was incubated for 45 minutes. Cells were visualized on a Leica TCS SP2 AOBS confocal laser scanning microscope. All measurements were performed by two independent investigators in a double-blind manner.

[0523] SPQ intracellular chloride concentration determination. The transport activity of ΔF508-CFTR activity in CFBE cells was assessed using the SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium) assay, because the fluorescence of SPQ is negatively correlated with increasing concentrations of intracellular chloride (Illsley, NP and Verkman, AS. Biochemistry 1987, 26, 1215). CFBE cells were grown on 96-well plates pre-coated with 1 mg / ml human fibronectin, 3 mg / ml bovine collagen I and 1 mg / ml bovine serum albumin using DMEM medium supplemented with L-glutamine and 10% FBS. Cells were first treated for 24 hours in the presence or absence of 20 μM CFTR calibrant VX809 (Van Goor, F et al. Proc. Natl. Acad. Sci. USA 2011, 108, 18843) and 1 hour with 50 μM CAL-PDZ domain inhibitor. The cells were then loaded with SPQ using a hypotonic shock for 15 minutes at 37°C using 10 mM SPQ in a 1:1 (v / v) Opti-MEM / water solution. The cells were then washed and incubated twice for 10 minutes with fluorescence quenching NaI buffer (130 mM NaI, 5 mM KNO3, 2.5 mM Ca(NO3)2, 2.5 mM Mg(NO3)2, 10 mM D-glucose, 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES, pH 7.4)). Subsequently, the cells were switched to a dequenching isotonic NaNO3 buffer (same as NaI buffer, except that 130 mM NaI was replaced by 130 mM NaNO3) with a CFTR activation cocktail (10 μM forskolin and 50 μM genistein). The fluorescence of iodide effluent mediated by CFTR was measured by incubating cells with activation mixture and CFTR specific inhibitor GlyH101 (10 μM). The effect of CAL-PDZ inhibitor was evaluated by the fluorescence increase rate above the basal level. The fluorescence of SPQ dequenched was measured using a plate reader VICTOR X3 (Perkin Elmer) with an excitation wavelength at 350nm and a DAPI emission filter. Data are expressed as mean ± standard deviation from at least three independent experiments.

[0524] A co-cleaved amphiphilic cyclic peptide ring (FΦRRRRQ) (cFΦR4) has been reported as a highly active cell penetrating peptide (CPP) that can enter the cytoplasm of mammalian cells through endocytosis and endosomal escape (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). In order to test the effectiveness of the reversible cyclization strategy, an N-3-mercaptopropionyl-FΦRRRRCK-NH2 peptide was synthesized and then cyclized by forming an intramolecular disulfide bond (Figure 17; Table 10, peptide 1). A linear peptide of the same sequence was also synthesized by replacing the N-terminal 3-mercaptopropionyl group with a butyryl group and replacing the C-terminal cysteine ​​with 2-aminobutyric acid (Abu or U) (Table 10, peptide 2). Both peptides were labeled with fluorescein isothiocyanate (FITC) at the C-terminal lysine residue, and their cellular uptake was evaluated by live cell confocal microscopy and flow cytometry. HeLa cells treated with cyclotide (5 μM) showed strong diffuse green fluorescence throughout the cell volume, while the endocytosis marker rhodamine-labeled dextran (dextran) Rho ) only showed punctate fluorescence in the cytoplasm ( Fig.18A ). The almost uniform distribution of FITC fluorescence in the cytoplasmic and nuclear regions indicated that the cyclic peptide was effectively internalized by HeLa cells and was able to escape from endosomes as efficiently as the parent cyclic peptide cFΦR4. In contrast, cells treated with the linear control peptide showed much weaker intracellular fluorescence under the same imaging conditions. Quantification of total intracellular fluorescence by fluorescence activated cell sorting (FACS) gave mean fluorescence intensities (MFI) of 27,100, 5530, and 1200 arbitrary units (AU) for cells treated with the disulfide cyclic peptide, the linear peptide, and FITC alone, respectively ( Fig.18B). The highly negatively charged pentapeptide Asp-Glu-pCAP-Leu-Ile (PCP, where pCAP is phospho-coumarylaminopropionic acid) was also used as a cargo and was linked to peptides 1 and 2 via a polyethylene glycol linker (Figure 17). pCAP is non-fluorescent, but when delivered to the mammalian cytoplasm, it undergoes rapid dephosphorylation to produce the fluorescent product coumaroylaminopropionic acid (CAP) (Stanford, SM et al. Proc. Natl. Acad. Sci. USA 2012, 109, 13972). Therefore, the pCAP assay provides a quantitative assessment of the cytoplasmic / nuclear concentration of different CPPs (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). FACS analysis of HeLa cells treated with 5 μM peptide 1-PCP and peptide 2-PCP resulted in MFI values ​​of 3020 and 700, respectively (Figure 19). Therefore, the above results indicate that the cyclization of FΦ RRRR through disulfide bonds can have similar effects as N- to -C cyclization and can increase its cellular uptake efficiency by about 5 times (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). In addition, cyclization through disulfide bond formation can enhance the proteolytic resistance of peptides. Incubation of peptide 1 with a protease mixture for 12 hours resulted in <50% degradation, while linear peptide 2 was degraded with a half-life of about 20 minutes under the same conditions ( Fig. 20 ).

[0525] Table 10. Sequences of peptides.

[0526]

[0527] a Amc, 7-amino-4-methylcoumarin; FITC, fluorescein isothiocyanate; Φ, L-2-naphthylalanine; Ω, norleucine; U, 2-aminobutyric acid.

[0528] To illustrate the utility of the reversible cyclization strategy, it was used to deliver specific caspase substrates into cells and monitor intracellular caspase activity in real time (Riedl, SJ and Shi, Y. Nat. Rev. Mol. Cell Biol. 2004, 5, 897). Although peptidyl coumarin derivatives have been widely used to detect caspase activity in vitro (Maly, DJ et al. Chembiochem 2002, 3, 16), they are generally not suitable for in vivo applications due to the impermeability of mammalian cell membranes. To generate a cell permeable caspase substrate, the caspase 3 / 7 substrate Ac-Asp-Nle-Abu-Asp-Amc (Thornberry, NA et al. J. Biol. Chem. 1997, 272, 17907) (Table 10, peptide 3, wherein Amc is 7-amino-4-methylcoumarin and Nle is norleucine) was fused to the CPP motif RRRRΦF. The fusion peptide was then cyclized to give cyclic peptide 4 (Table 10) by adding a 3-mercaptopropionyl group to its N-terminus, replacing the C-terminal Abu with cysteine, and forming an intramolecular disulfide bond. For comparison, an isomeric but irreversibly cyclized peptide (Table 10, peptide 5) was synthesized by forming a thioether bond between the N-terminal bromobutyryl moiety and the C-terminal cysteine ​​(Figure 17). A linear control peptide of the same sequence (Table 10 peptide 6) was also prepared as described above. Finally, the caspase 3 / 7 substrate was conjugated to a non-arginine (R9) to generate a positive control peptide (Table 10, peptide 7). In vitro kinetic analysis revealed that fusion of the caspase 3 / 7 substrate to RRRRΦF and R9 reduced activity by 53% and 72% relative to peptide 3, respectively, while cyclization by thioether formation inactivated the peptide against recombinant caspase 3 (Table 11). The activity of peptide 4 against caspase 3 could not be reliably determined because caspase assays require a reducing environment to cleave disulfide bonds. Given the structural similarity between peptides 4 and 5, it can be assumed that the cyclic form of peptide 4 is also inactive against caspases, but has similar activity to peptide 6 after reductive cleavage of the disulfide bond.

[0529] Table 11. In vitro activity of various fluorogenic substrates on recombinant caspase-3 enzyme.

[0530]

[0531] Jurkat cells were pretreated with the kinase inhibitor staurosporine to induce caspase activity and thus apoptosis (Belmokhtar, CA et al. Biochem. J. 1996, 315, 21). These cells were then incubated with peptides 3-7 and the amount of Amc released was monitored at different time points (0-10 hours). The non-permeable caspase substrate (peptide 3) produced almost no fluorescence increase over a 10-hour period ( Fig.21 ). Peptide 4 produced the fastest fluorescence increase, reaching 459 fluorescence units (FU), followed by peptides 7 and 6. Peptide 5, which is inactive against caspase 3, also produced AMC in a time-dependent manner, although at a much slower rate (99 FU). This slow rate of AMC release can be attributed to hydrolysis by other intracellular proteases and peptidases. Consistent with this explanation, pretreatment of Jurkat cells with the trypsin inhibitor Z-VAD (OMe) -FMK (Slee, EA et al. Biochem J. 1996, 315, 21) and then incubation with peptide 4 released AMC at a rate similar to that of peptide 5 alone. One explanation for the above observations is that both peptides 4 and 5 can effectively enter the interior of the cell, but only peptide 4 can be converted into a linear caspase substrate within the cell.

[0532] Many protein-protein interactions (PPIs) are mediated by protein domains that bind short peptides in their extended conformations (e.g., α-helices and β-strands) (Pawson, T and Nash, P. Science 2003, 300, 445). For example, the PDZ domain is a common structural domain of 80-90 amino acids found in bacterial to human signaling proteins (Doyle, DA et al. Cell 1996, 85, 1067; Morais Cabral, JH et al. Nature 1996, 382, ​​649; Lee, HJ and Zheng, JJ. Cell Commun. Signal. 2010, 8, 8). The PDZ domain recognizes a specific sequence at the C-terminus of its binding partner, and the bound peptide ligand is in its extended β-strand conformation (Doyle, DA et al. Cell 1996, 85, 1067; Songyang, Z et al. Science 1997, 275, 73). 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). Previous library screening and rational design have identified several moderately potent peptide-based inhibitors of the CAL-PDZ domain (K D Values ​​range from high nM to low μM) (Cushing, PR et al. Angew. Chem. Int. Ed. 2010, 49, 9907; Roberts, KE et al. PLos Comput. Biol. 2008, 8, e1002477; Kundu, R et al. Angew. Chem. Int. Ed. 2012, 51, 7217-7220). However, none of the peptide inhibitors are cell permeable, which limits their therapeutic potential.

[0533] Starting from the hexapeptide ligand WQVTRV of the CAL-PDZ domain (Roberts, KE et al. PLos Comput. Biol. 2008, 8, e1002477), a disulfide bond-mediated cyclic peptide was designed by adding the sequence CRRRRF to its N-terminus and replacing the Val at position -3 with cysteine ​​(Table 10, peptide 8). Thus, in peptide 8, the tryptophan residue at position -5 was designed to have dual functions for PDZ binding and membrane translocation. To facilitate affinity measurements and quantification of its cellular uptake, a FITC group was added to the N-terminus of peptide 8. FA analysis showed that in the absence of a reducing agent, peptide 8 showed no detectable binding to the CAL-PDZ domain ( Fig.22A In the presence of 2 mM tris(carboxyethyl)phosphine, which can reduce disulfide bonds, peptide 8 was synthesized with a K of 489 nM. D The value is bound to the CAL-PDZ domain. Peptide 8 easily permeabilizes cells; incubation of HeLa cells with 5 μM peptide 8 for 2 hours resulted in strong and diffuse fluorescence throughout the cells ( Fig. 22B ).

[0534] As expected, peptide 8 readily permeabilized cells ( Fig.25 C). Bronchial epithelial CFBE cells homozygous for the ΔF508-CFTR mutation were treated with 10 μM Corr-4a in the presence and absence of 50 μM unlabeled peptide 8. Peptide 8 is expected to increase the amount of ΔF508-CFTR protein transferred to the plasma membrane by inhibiting the function of the CAL-PDZ domain, while Corr-4a is a small molecule that helps the folding of the ΔF508-CFTR protein delivered to the plasma membrane. Immunostaining of untreated cells ( Fig.25 D, Figure I) shows that most of the expressed ΔF508-CFTR is in the endoplasmic reticulum surrounding the nucleus. In contrast, cells treated with Corr-4a and peptide 8 produced much greater amounts of protein on the cell surface ( Fig.25 D, Panel II). Quantification of the cell population revealed that a small but significant percentage of cells had a wild-type-like distribution of ΔF508-CFTR on the cell surface ( Fig.25 D). Finally, the SPQ assay was used to quantify the ion channel activity of ΔF508-CFTR CFBE cells that were either untreated or treated with the CTFR folding corrector VX809 and peptide 8. Again, VX809 and peptide 8 acted synergistically to improve the function of the channel activity of ΔF508-CFTR ( Fig.25 E).

[0535] Example 3

[0536] Cyclic peptides have great potential as therapeutic agents and research tools, but are generally impermeable to cell membranes. Fusion of cyclic peptides with cyclic cell-penetrating peptides can generate bicyclic peptides that can be cell-permeable and can retain the ability to recognize specific intracellular targets. Application of this strategy to protein tyrosine phosphatase 1B and peptidylprolyl cis-trans isomerase Pin1 resulted in potent, selective, proteolytically stable and biologically active inhibitors against the enzymes.

[0537] Cyclic peptides (and depsipeptides) exhibit a wide range of biological activities (Pomilio, AB et al. Curr. Org. Chem. 2006, 10, 2075-2121). Several innovative methods have been developed recently to synthesize cyclic peptides, individually (Meutermans, WDF et al. J.Am.Chem.Soc. 1999, 121, 9790-9796; Schafmeister, CE et al. J.Am.Chem.Soc. 2000, 122, 5891-5892; Sun, Y et al. Org.Lett. 2001, 3, 1681-1684; Kohli, RM et al. Nature 2002, 418, 658-661; Qin, C et al. J.Comb.Chem. 2004, 6, 398-406; Turner, RA et al. Org.Lett. 2007, 9, 5011-5014; Hili, R et al. J.Am.Chem. Soc. 2010, 132, 2889-2891; Lee, J et al. J. Am. Chem. Soc. 2009, 131, 2122-2124; Frost, JR et al. ChemBioChem 2013, 14, 147-16) or in combination (Eichler, J et al. Mol. Divers. 1996, 1, 233-240; Giebel, LB et al. Biochemistry 1995, 34, 15430-15435; Scott, CP et al. Proc. Natl. Acad. Sci. USA 1999, 96, 13638-13643; Millward, SW et al. J. Am. Chem. Soc. 2005, 127, 14142-14143; Sako, Y et al. J. Am. Chem. Soc. 2008, 130, 7232-7234.; Li, S et al. Chem. Commun. 2005, 581-583.; Joo, SH et al. J. Am. Chem. Soc. 2006, 128, 13000-13009; Heinis, C et al. Nat. Chem. Biol. 2009, 5, 502-507; Tse, BN et al. J. Am. Chem. Soc. 2008, 130, 15611-15626), and screened their biological activities.A particularly exciting application of cyclic peptides is the inhibition of protein-protein interactions (PPIs) (Leduc, AM et al. Proc. Natl. Acad. Sic. USA 2003, 100, 11273-11278; Millward, SW et al. ACS Chem Biol 2007, 2, 625-634; Tavassoli, A et al. ACS Chem. Biol. 2008, 3, 757-764.; Wu, X et al. Med. Chem. Commun. 2013, 4, 378-382; Birts, CN et al. Chem. Sci. 2013, 4, 3046-3057; Kawakami, T et al. ACS Chem. Biol. 2013, 8, 1205-1214; Lian, W et al. J.Am.Chem.Soc.2013,135,11990-11995), it remains a challenging target for conventional small molecules. However, the main limitation of cyclic peptides is that they are generally unable to permeate cell membranes, excluding any application for intracellular targets, which include most therapeutically relevant PPIs. Despite the formation of intramolecular hydrogen bonds (Rezai, T et al. J.Am.Chem.Soc.2006,128, 14073-14080) or the N of the peptide backbone. α -Methylation (Chatterjee, J et al. Acc. Chem. Res. 2008, 41, 1331-1342; White, TR et al. Nat. Chem. Biol. 2011, 7, 810-817) can improve the membrane permeability of certain cyclic peptides, but alternative strategies to increase the cell permeability of cyclic peptides are clearly needed.

[0538] Protein tyrosine phosphatase 1B (PTP1B) is a prototype member of the PTP superfamily and plays many roles during eukaryotic cell signaling. Due to its role in negatively regulating insulin and leptin receptor signaling, PTP1B is an effective target for treating type II diabetes and obesity (Elchelby, M et al. Science 1999, 283, 1544–1548; Zabolotny, JM et al. Dev. Cell 2002, 2, 489-495). A large number of PTP1B inhibitors have been reported (He, R et al. New Therapeutic Strategies for Type 2 Diabetes: Small Molecule Approaches. RM Jones, RSC Publishing 2012, p. 142), but none of them have been successful in the clinic. Designing PTP inhibitors is challenging because most phosphotyrosine (pY) isosteres such as difluorophosphonomethylphenylalanine (F2Pmp) (Burke Jr., TR et al. Biochem. Biophys. Res. Commun. 1994, 204, 129-134) cannot penetrate cell membranes. In addition, because all PTPs share similar active sites, it is difficult to achieve selectivity for a single PTP. In this article, a potential general method for designing cell-permeable cyclic peptide-based inhibitors for intracellular proteins (e.g., PTP1B) is reported.

[0539] Materials. Fmoc-protected amino acids were purchased from Advanced ChemTech (Louisville, KY), Peptides International (Louisville, KY), or Aapptec (Louisville, KY). Fmoc-F2Pmp-OH was purchased from EMD Millipore (Darmstadt, Germany). Aminomethyl-ChemMatrix resin (0.66 mmol / g) was from SJPC (Quebec, Canada). Rink resin LS (100-200 mesh, 0.2 mmol / g) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) were purchased from Advanced ChemTech. O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 1-hydroxybenzotriazole hydrate (HOBt) were purchased from Aapptec. Phenyl isothiocyanate, fluorescein isothiocyanate (FITC), and rhodamine B-labeled dextran (dextran) in 1-mL sealed ampoules were purchased from Aapptec. Rho ) were purchased from Sigma-Aldrich. Cell culture medium, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-EDTA, Dulbecco's phosphate-buffered saline (DPBS) (2.67 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 8.06 mM sodium dihydrogen phosphate), and anti-phospho-IR / IGF1R antibody were purchased from Invitrogen (Carlsbad, CA). Nuclear staining dye DRAQ5 TM Antibodies to β-actin and β-actin were purchased from Thermo Scientific (Rockford, IL). Antibody 4G10 was purchased from Millipore (Temecula, CA). All solvents and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification unless otherwise noted.

[0540] Cell culture A549, HEK293 and HepG2 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS in a humidified incubator at 37°C with 5% CO2.

[0541] Protein expression, purification and labeling. The gene encoding the catalytic domain of PTP1B (amino acids 1-321) was amplified by polymerase chain reaction using PTP1B cDNA as a template and oligonucleotides 5'-ggaattccatatggagatggaaaaggagttcgagcag-3' and 5'-gggatccgtcgacattgtgtggctccaggattcgtttgg-3' as primers. The resulting DNA fragment was digested with endonucleases Nde I and Sal I and inserted into the prokaryotic vector pET-22b (+)-ybbR (Yin, J et al. Proc. Natl. Acad. Sci. USA 2005, 102, 15815-1582 0). The cloning procedure resulted in the addition of a ybbR tag (VLDSLEF IASKL) to the N-terminus of PTP1B. Expression and purification of ybbR-labeled PTP1B were performed as previously described (Ren, L et al. Biochemistry 2011, 50, 2339-2356). PTP1B was labeled with Texas Red (Yin, J et al. Proc. Natl. Acad. Sci. USA 2005, 102, 15815-15820) by treating ybbR-labeled PTP1B protein (80 μM) in 50 mM HEPES, pH 7.4, 10 mM MgCl2 with Sfp phosphopantetheine transferase (1 μM) and Texas Red-CoA (100 μM) for 30 minutes at room temperature. The reaction mixture was passed through a G-25 fast desalting column balanced in 30 mM HEPES, pH 7.4, 150 mM NaCl to remove any free dye molecules. Full-length human S16A / Y23A mutant Pin1 was expressed and purified from E. coli as previously described (Liu, T et al. JM ed. Chem. 2010, 53, 2494-2501).

[0542] Library synthesis. The cyclic peptide library was synthesized on 1.35 g of aminomethyl-ChemMatrix resin (0.57 mmol / g). Unless otherwise stated, library synthesis was performed at room temperature. The linker sequence (BBM) was synthesized using standard Fmoc chemistry. A typical coupling reaction contained 5 equivalents of Fmoc-amino acid, 5 equivalents of HBTU, and 10 equivalents of diisopropylethylamine (DIPEA), and was allowed to continue mixing for 2 hours. The Fmoc group was removed by treating twice (5+15 minutes) with 20% (v / v) piperidine in DMF, and the beads were washed thoroughly with DMF (6×). In order to spatially separate the beads into an outer layer and an inner layer, the resin (after removing the N-terminal Fmoc group) was washed with DMF and water and soaked in water overnight. The resin was quickly drained and suspended in a solution of Fmoc-Glu (δ-NHS)-OA11 (0.10 equivalents), Boc-Met-OSu (0.4 equivalents) and N-methylmorpholine (2 equivalents) in 20 mL 1:1 (v / v) DCM / diethyl ether (Joo, SH et al. J. Am. Chem. Soc. 2006, 128, 13000-13009). The mixture was incubated on a rotary shaker for 30 minutes. The beads were washed with 1:1 DCM / diethyl ether (3×) and DMF (8×). Next, the Fmoc group was removed by piperidine treatment. Then, Fmoc-Arg (Pbf) -OH (4×), Fmoc-Nal-OH and Fmoc-Phe-OH were sequentially coupled to half of the resin by standard Fmoc chemistry. The other half was coupled with the same amino acid in reverse sequence. The resins were combined and random sequences were synthesized by the split-and-pool method using 5 equivalents of Fmoc-amino acid, 5 equivalents of HATU and 10 equivalents of DIPEA as coupling agents. The coupling reaction was repeated once to ensure complete coupling at each step. For random positions, a 24-amino acid set was selected based on their structural diversity, metabolic stability, and commercial availability, including 10 proteinogenic α-L-amino acids (Ala, Asp, Gln, Gly, His, Ile, Ser, Trp, Pro, and Tyr), 5 non-proteinogenic α-L-amino acids (L-4-fluorophenylalanine (Fpa), L-homoproline (Pip), L-norleucine (Nle), L-phenylglycine (Phg), and L-4-(phosphonodifluorophenylamino)phenylalanine (F2Pmp)), and 9 α-D-amino acids (D-2-naphthylalanine (D-Nal), D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, D-Pro, D-Thr, and D-Val).In order to distinguish isobaric amino acids during PED-MS analysis, 4% (mol / mol) of CD3CO2D was added to the coupling reactions of D-Ala, D-Leu and D-Pro, while 4% CD3CD2CO2D was added to the Nle reaction. Fmoc-F2Pmp-OH (0.06 eq.) and Fmoc-Tyr-OH (0.54 eq.) were placed in the middle of random positions using HATU / DIPEA. After the entire sequence was synthesized, the allyl group on the C-terminal Glu residue was removed (3×) by treatment with a DCM solution containing tetrakis(triphenylphosphine)palladium [Pd(PPh3)4, 0.25 eq.] and phenylsilane (5 eq.) for 15 minutes. The beads were washed sequentially with 0.5% (v / v) DIPEA in DMF, 0.5% (w / v) sodium dimethyldithiocarbamate hydrate in DMF, DMF (3×), DCM (3×), and DMF (3×). The Fmoc group on the N-terminal random residue was removed with piperidine as described above. The beads were washed with DMF (6×), DCM (3×), and 1 M HOBt in DMF (3×). For peptide cyclization, a solution of PyBOP / HOBt / DIPEA (5, 5, 10 equiv., respectively) in DMF was mixed with the resin, and the mixture was incubated on a rotary shaker for 3 h. The resin was washed with DMF (3×) and DCM (3×) and dried under vacuum for >1 h. Side chain deprotection was performed with modification reagent K (78.5:7.5:5:5:2.5:1:1 (v / v) TFA / phenol / water / thioanisole / ethanedithiol / anisole / triisopropylsilane) for 3 hours. The resin was washed with TFA and DCM and dried under vacuum and then stored at -20°C.

[0543] Library screening and peptide sequencing. The library resin (100 mg, about 300,000 beads) was swollen in DCM, washed thoroughly with DMF, deionized water H2O, and incubated for 3 hours at 4 ° C in 1 mL of a blocking buffer (PBS, pH 7.4, 150 mM NaCl, 0.05% Tween 20 and 0.1% gelatin) containing 20 nM Texas Red-labeled PTP1B. The beads were examined under an Olympus SZX12 microscope equipped with a fluorescent illuminator (Olympus America, Center Valley, PA), and the strongest fluorescent beads were manually collected as positive hits. The beads containing the encoded linear peptides were sequenced separately by partial Edman degradation-mass spectrometry (PED-MS) (Liu, T et al. J. Med. Chem. 2010, 53, 2494-2501).

[0544] Single peptide synthesis and labeling. Single and double-ring peptides were synthesized on Rink Resin LS (0.2 mmol / g) using standard Fmoc chemistry. For single-ring peptides, after coupling the last (N-terminal) residue, the allyl group on the C-terminal Glu residue was removed by treatment with Pd(PPh3)4 and phenylsilane (0.1 and 10 equivalents, respectively) in anhydrous DCM (3×15 minutes). The N-terminal Fmoc group was removed by treatment with 20% (v / v) piperidine in DMF, and the peptide was cyclized by treatment with PyBOP / HOBt / DIPEA (5, 5, and 10 equivalents) in DMF for 3 hours. For double-ring peptides, the N-terminal Fmoc group was removed with piperidine, and trimesic acid was coupled to the N-terminal amine using HBTU as a coupling agent. The allyloxycarbonyl groups on the side chains of the two Dap residues were removed by treatment with Pd(PPh3)4 and phenylsilane (0.1 and 10 equivalents, respectively) in anhydrous DCM for 2 hours. The resulting peptide was cyclized with PyBOP as described above. The peptide was deprotected and released from the resin by treatment with 82.5:5:5:5:2.5 (v / v) TFA / thioanisole / water / phenol / ethanedithiol for 2 hours. The peptide was triturated with cold ether (3×) and heated at C 18 The peptides were purified by reverse phase HPLC on the column. The authenticity of each peptide was confirmed by MALDI-TOF mass spectrometry. The peptides were labeled with FITC by dissolving the purified peptides (approximately 1 mg) in 300 μL of 1:1:1 (vol / vol) DMSO / DMF / 150 mM sodium bicarbonate (pH 8.5) and mixing with 10 μL of FITC in DMSO (100 mg / mL). After 20 minutes at room temperature, the peptides were incubated at C 18 The reaction mixture was subjected to reverse phase HPLC on a column to separate the FITC-labeled peptide.

[0545] PTP inhibition assay. PTP assays were performed in a quartz microcuvette (total volume 150 μL). The reaction mixture contained 100 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2 mM EDTA, 1 mM TCEP, 0-1 μM PTP inhibitor and 500 μM p-nitrophenyl phosphate (pNPP). The enzymatic reaction was started by adding PTP (final concentration 15-75 nM) and continuously monitored at 405 nm on a UV-VIS spectrophotometer. The initial rate was calculated from the reaction progress curve (usually < 60 seconds). The half maximal inhibition constant (IC 50 ) is defined as the concentration of inhibitor that reduces enzyme activity to 50% and is obtained by plotting the rate (V) versus inhibitor concentration [I] and fitting the data to the following equation:

[0546]

[0547] Where V0 is the enzyme reaction rate in the absence of inhibitor. The inhibition constant (K) was determined by measuring the initial rate at a fixed enzyme concentration (15 nM) but varying concentrations of pNPP (0-24 mM) and inhibitor (0-112 nM). i ). The reaction rate (V) was plotted against the pNPP concentration ([S]) and fitted according to the following equation:

[0548]

[0549] To obtain the Michaelis constant K. K was obtained by plotting the K value against the inhibitor concentration [I] and fitting it to the following equation: i value:

[0550]

[0551] where K0 is the Michaelis constant in the absence of an inhibitor ([I] = 0).

[0552] Confocal microscopy. Approximately 5×10 4 A549 cells were seeded in 35-mm glass-bottom microwell dishes (MatTek) containing 1 mL of culture medium and cultured for 1 day. A549 cells were gently washed once with DPBS and incubated with FITC-labeled PTP1B inhibitor (5 μM), dextran, and 1 μM cytotoxicity factor (TGF-β) in the presence of 5% CO2. Rho (1mg mL -1 ) in growth medium at 37°C for 2 hours. The peptide-containing medium was removed, and the cells were washed three times with DPBS and incubated in 1 mL of DPBS containing 5 μM DRAQ5 for 10 minutes. The cells were washed twice again with DPBS. The cells were then imaged on a Visitech Infinity 3Hawk2D-array live cell imaging confocal microscope (with a 60× oil immersion lens) at 37°C in the presence of 5% CO2. Live cell confocal microscopy imaging of HEK293 cells treated with FITC-labeled Pin1 inhibitors was performed similarly.

[0553] Immunoblotting. A549 cells were cultured in complete growth medium to reach 80% confluence. Cells were starved for 3 hours in serum-free medium and treated with different concentrations of PTP1B inhibitor for 2 hours, followed by incubation in medium supplemented with 1 mM sodium pervanadate for 30 minutes. The solution was removed and the cells were washed twice with cold DPBS. Cells were detached and lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 10 mM sodium pyrophosphate, 5 mM iodoacetic acid, 10 mM NaF, 1 mM EDTA, 2 mM sodium pervanadate, 0.1 mg / mL phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 0.1 mg / mL trypsin inhibitor. After incubation on ice for 30 minutes, the cell lysate was centrifuged at 15,000 rpm in a microcentrifuge for 25 minutes. Total cellular proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes and immunoblotted using the anti-phosphotyrosine antibody 4G10. The same samples were analyzed on separate SDS-PAGE gels and stained with Coomassie Brilliant Blue to ensure equal sample loading in all lanes.

[0554] To test the effect of inhibitors on the insulin signaling pathway, HepG2 cells were cultured to reach 80% confluence. Cells were starved for 4 hours in serum-free DMEM medium, then treated with PTP1B inhibitors (2 hours) and then stimulated with 100 nM insulin for 5 minutes. Samples were analyzed by SDS-PAGE as described above and immunoblotted using anti-phospho-IR / IGF1R antibodies. PVDF membranes were also probed with anti-β-actin antibodies as loading controls.

[0555] Serum stability test. Stability test was performed by modifying a previously reported procedure (Nguyen, LT et al. PLoS One, 2010, 5, e12684). Diluted human serum (25%) was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected. The peptide stock solution was diluted into the supernatant to a final concentration of 5 μM and incubated at 37°C. At various time points (0-24 hours), a 200-μL aliquot was taken out and mixed with 50 μL of 15% trichloroacetic acid and incubated overnight at 4°C. The final mixture was centrifuged at 15,000 rpm for 10 minutes in a microcentrifuge and passed through a 400 μL centrifuge equipped with C 18 The supernatant was analyzed by reverse phase HPLC of the column. The amount (%) of remaining peptide was determined by integrating the area under the peptide peak (monitored at 214 nm) and compared with that of a control reaction (without serum).

[0556] Fluorescence anisotropy. FA experiments were performed by incubating 100 nM FITC-labeled peptides with different concentrations of protein in 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM magnesium acetate, and 0.1% bovine serum albumin (BSA) for 2 hours at room temperature. FA values ​​were measured on a Molecular Devices Spectramax M5 plate reader with excitation and emission wavelengths of 485 nm and 525 nm, respectively. The equilibrium dissociation constant (K) was determined by plotting the FA values ​​as a function of protein concentration and fitting the curve to the following equation: D ):

[0557]

[0558] where Y is the FA value at a given protein concentration x; L is the peptide concentration; Q b / Q f is the correction factor for fluorophore-protein interaction; A 最大值 is the maximum FA when all peptides are bound to the protein, and A 最小值 is the minimum FA value when all peptides are free. FA competition assays were performed by incubating 100 nM FITC-labeled Pin1 inhibitor 5 with 1 μM Pin1 and then adding 0-5 μM unlabeled inhibitor. FA values ​​were similarly measured on a plate reader. IC was obtained by plotting FA values ​​against competitor concentration and curve fitting using a four-parameter dose-response inhibition equation. 50 Values ​​(Prism 6, GraphPad).

[0559] Recently, a class of cell penetrating peptides (CPPs), cyclo(Phe-Nal-Arg-Arg-Arg- Arg-Gln) (cFΦR4, where Φ or Nal is L-naphthylalanine) was discovered (Qian, Z et al. A CS Chem. Biol. 2013, 8, 423-431). Unlike previous CPPs, which are usually linear peptides and mainly trapped in endosomes, cFΦR4 can effectively escape from endosomes into the cytoplasm. Short peptide cargoes (1-7aa) can be delivered to mammalian cells by direct incorporation into the cFΦR4 ring. The possibility of developing bifunctional cyclic peptides containing both cell permeability and target binding sequences as cell permeability inhibitors against intracellular proteins was studied. To generate specific inhibitors against PTP1B, a single-bead two-compound library was synthesized on a spatially separated ChemMatrix resin (Liu, R et al. J. Am. Chem. Soc. 2002, 124, 7678-7680), where each bead displays a bifunctional cyclic peptide on its surface and contains the corresponding linear peptide as a coding tag in its interior ( Fig.23 and Fig.24 The bifunctional cyclic peptides are characterized by having an amphiphilic CPP motif FΦR4 (or its reverse sequence RRRR ΦF) on one side and a random pentapeptide sequence (X 1 X 2 X 3 X 4 X 5 ), where X 2 represents a 9:1 (mol / mol) mixture of Tyr and F2Pmp, while X 1 and X 3 -X 5 It is any one of the following 24 amino acids: 10 proteinogenic L-amino acids (Ala, Asp, Gln, Gly, His, Ile, Pro, Ser, Tyr, Trp), 5 unnatural α-L-amino acids (F2Pmp, L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-phenylglycine (Phg), L-piperidinic acid (Pip)) and 9 α-D-amino acids (D-Ala, D-Asn, D-Glu, D-Leu, L-β-naphthylalanine (D-Nal), D-Phe, D-Pro, D-Thr and D-Val). 2 The use of a 9:1 Tyr / F2Pmp ratio together with a 5-fold reduction in surface peptide loading reduced the amount of F2Pmp-containing peptides at the bead surface by 50-fold, thereby increasing stringency and minimizing nonspecific binding during library screening (Chen, X et al. J. Comb. Chem. 2009, 11, 604-611). Library against Texas Red-labeled PTP1B (theoretical diversity 6. 6×10 5 ) generated 65 positive beads, which were individually sequenced by partial Edman degradation-mass spectrometry (PED-MS) (Thakkar, A et al. Anal. Chem. 2006, 78, 5935-5939), resulting in 42 complete sequences (Table 12). Interestingly, most of the selected PT P1B inhibitors contain an inverted CPP motif (RRRRΦF).

[0560] Table 12. Peptide sequences selected from a cyclic peptide library targeting PTP1B a

[0561]

[0562]

[0563] aFpa, L-4-fluorophenylalanine; Pip, L-homoproline; Nle, L-norleucine; Phg, L-phenylglycine; F2Pmp, L-4-(phosphonodifluoromethyl)phenylalanine.

[0564] *Sequences subjected to further analysis.

[0565] Three hit sequences (D-Thr-D-Asn-D-Val-F2Pmp-D-Ala-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 1), Ser-D-Val-Pro-F2Pmp-His-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 2), and Ile-Pro-Phg-F2Pmp-Nle-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 3)) were resynthesized and purified by HPLC. All three peptides were competitive PTP1B inhibitors (Table 13), with peptide 2 being the most potent (K I =5 4nM)( Fig.25 Confocal microscopy analysis of human cells treated with fluorescein isothiocyanate (FITC)-labeled inhibitor 2 showed poor cellular uptake of the peptide ( Fig.26 a). It has been previously shown that the cellular uptake efficiency of cyclic peptides decreases as the size of the cargo inserted into the cFΦR4 loop increases (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423-431). Larger loops may be more conformationally flexible and may bind less tightly to cell surface receptors (e.g., membrane phospholipids) during endocytosis. Negatively charged F2Pmps may also interact intramolecularly with the FΦR4 motif and interfere with its CPP function.

[0566] Table 13. Efficacy of selected monocyclic peptide inhibitors against PTP1B

[0567]

[0568] To improve the cell permeability of inhibitor 2, a bicyclic system in which the CPP motif is located in one ring and the target binding sequence constitutes the other ring was explored ( Fig.23). The bicyclic system keeps the CPP ring to a minimum size, which can lead to more efficient cellular uptake according to previously observed trends (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423-431). The bicyclic system should be able to accommodate cargoes of any size, since the incorporation of the latter does not change the size of the CPP ring, and therefore should not affect the delivery efficiency of the cyclic CPP. The use of a rigid scaffold (e.g., trimesic acid) can also help keep the CPP and cargo motifs away from each other and minimize any mutual interference. Compared to its monocyclic counterpart, the smaller ring of the bicyclic peptide can lead to greater structural rigidity and improved metabolic stability.

[0569] To convert the monocyclic PTP1B inhibitor 2 into a bicyclic peptide, the Gln residue (for attachment to the solid support and peptide cyclization) was replaced with (S)-2,3-diaminopropionic acid (Dap), and a second Dap residue was inserted at the junction of the CPP and PTP1B binding sequences (C-terminal to His) ( Fig.23 The synthesis of the bicyclic ring is completed by forming three amide bonds between trimesic acid and the N-terminal amine and the side chains of two Dap residues ( Fig. 27 ) (Lian, W et al. J. Am. Chem. Soc. 2013, 135, 11990-11995). Briefly, standard Fmoc chemistry and N β -Allyloxycarbonyl (Alloc) protected Dap was synthesized as a linear peptide on Rink amide resin. After removal of the N-terminal Fmoc group, the exposed amine was acylated with trimesic acid. Removal of the Alloc group with Pd(PPh3)4 followed by treatment with PyBOP gave the desired bicyclic structure. Lysine was added to the C-terminus for ease of labeling with fluorescent probes. The bicyclic peptide (peptide 4) was deprotected with TFA and purified to homogeneity by HPLC.

[0570] Bicyclic peptide 4 can be used as a competitive inhibitor of PTP1B, and its K I The value was 37 nM (Figure 26b). It can be highly selective for PTP1B. When assayed against p-nitrophenyl phosphate as a substrate (500 μM), inhibitor 4 had IC values ​​of 30 and 500 nM for PTP1B and TCPTP, respectively. 50 value( Fig.26 c and Table 14). It exhibited minimal inhibition of any of the other PTPs tested (≤10% inhibition of HePTP, SHP-1, PTPRC, PTPH1, or PTPRO at 1 μM inhibitor concentration). Inhibitor 4 had improved cell permeability over peptide 2 as detected by live cell confocal microscopy of A549 cells treated with FITC-labeled inhibitor 4 ( Fig.26a). Treated cells showed diffuse fluorescence throughout the cytoplasm and nucleus as well as fluorescent spots, indicating that a portion of the inhibitor reached the cytoplasm and nucleus, while the rest was likely trapped in endosomes. Incubation of inhibitor 4 in human serum at 37°C for 24 h resulted in approximately 10% degradation, while 91% of inhibitor 2 was degraded under the same conditions ( Fig.28 In summary, inhibitor 4 is comparable to the small molecule PTP1B inhibitors reported so far in terms of potency, selectivity for the highly similar TCPTP (17-fold), cell permeability and stability (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423-431).

[0571] Table 14. Selectivity of bicyclic inhibitor 4 against various PTPs a

[0572]

[0573] a NA, no significant inhibition at 1 μM inhibitor.

[0574] Next, the ability of inhibitor 4 to interfere with PTP1B function during cell signaling was tested. Treatment of A549 cells with inhibitor 4 (0-5 μM) resulted in a dose-dependent increase in the phosphotyrosine (pY) levels of a large number of proteins, which is consistent with the broad substrate specificity of PTP1B (Ren, L et al. Biochemistry 2011, 50, 2339) (Figure 29a). Analysis of the same samples by Coomassie blue staining showed similar amounts of protein in all samples (Figure 29b), indicating that the pY levels increased reflected the phosphorylation (or reduced PTP reaction) of increase, rather than the change in total protein levels. Remarkably, the increase in tyrosine phosphorylation was already obvious under 8 nM inhibitor 4. Interestingly, the inhibitor concentration further increased more than 1 μM and reversed the effect on tyrosine phosphorylation, which was previously observed by Zhang and colleagues with different PTP1B inhibitors (Xie, L et al. Biochemistry 2003, 42, 12792-12804). To obtain further evidence that intracellular PTP1B is inhibited by peptide 4, we used a 1162 pY 1163Immunoblotting with site-specific antibodies was used to monitor the pY level of the insulin receptor (IR) (i.e., a clear PTP1B substrate in vivo) (Elchelby, M et al. Science 1999, 283, 1544–1548; Zabolotny, JM et al. Dev. Cell 2002, 2, 489-495). Again, treatment with inhibitor 4 caused a dose-dependent increase in insulin receptor phosphorylation up to 1 μM inhibitor, and the effect leveled off at higher concentrations (Figure 29c, d). In summary, these data indicate that the bicyclic inhibitor 4 can effectively enter mammalian cells and can inhibit PTP1B in vivo. The reduced phosphorylation at higher inhibitor concentrations may be caused by nonspecific inhibition of other PTPs (which can then downregulate protein tyrosine kinases). It may also reflect the pleiotropic effects exerted by PTP1B, which can negatively and positively regulate the activity of different protein kinases (Lessard, L et al. Biochim. Biophys. Acta 2010, 1804, 613).

[0575] To test the generality of the bicyclic approach, it was applied to design cell-permeable inhibitors against the peptidylprolyl cis-trans isomerase Pin1, a potential target for the treatment of a variety of human diseases including cancer (Lu, KP and Zhou, XZ. Nat. Rev. Mol. Cell Biol. 2007, 8, 904-916), for which potent, selective and bioactive inhibitors are still lacking (More, JD and Potter, A. Bioorg. Med. Chem. Lett. 2013, 23, 4283-91). Therefore, the previously reported single-ring peptide (5) is an effective in vitro inhibitor of Pin1 (K D 258nM), but is membrane impermeable (Liu, T et al. J Med. Chem. 2010, 53, 2494-2501), fused with cFΦR4 ( Fig.30 ). In addition, the L-Tyr at the pThr+3 position was replaced by Arg to improve water solubility. The resulting bicyclic peptide 6 binds to Pin1, and its K D The value was 131 nM (Table 15 and Fig.31 Insertion of D-Ala at the pThr+5 position to increase the separation between the Pin1 binding and cell-penetrating motifs increased inhibitor potency by approximately 2-fold (K of inhibitor 7 D =72 nM). Inhibitor 7 competed with FITC-labeled inhibitor 5 for binding to Pin1 (Figure 32), indicating that both can bind to the Pin1 active site. Substituting D-pThr of inhibitor 7 for D-pThr reduced its potency by about 10-fold (for inhibitor 8, KD =620 nM, Table 16), while further replacement of the piperoyl residue with D-Ala abolished Pin1 inhibitory activity (peptide 9). Bicyclic inhibitors 7-9 were cell permeable (Figure 33). Treatment of HeLa cells with inhibitor 7 resulted in a time- and dose-dependent inhibition of cell growth (45% inhibition after 3 days of treatment with 20 μM inhibitor 7), while monocyclic inhibitor 5 and inactive peptide 9 had no effect ( Fig.34 ). Peptide 8 also inhibited cell growth, but to a lesser extent than inhibitor 7.

[0576] Table 15. Dissociation constants of single- and double-cyclic peptides against Pin1 as determined by FA analysis

[0577]

[0578] a Dap, L-2,3-diaminopropionic acid; Nal, L-β-naphthylalanine; Pip, L-2-piperidinic acid; Sar, sarcosine; Tm, trimesic acid. For FA analysis, all peptides were labeled with FITC at the C-terminal lysine side chain.

[0579] In summary, a potential general approach to design cell-permeable bicyclic peptides against intracellular targets was developed. These preliminary studies showed that replacement of the PTP1B binding motif with other peptide sequences of different physicochemical properties also resulted in their effective delivery into cultured mammalian cells. The availability of a general intracellular delivery method should greatly expand the utility of cyclic peptides in drug development and biomedical research.

[0580] Example 4

[0581] The CPP sequences in Table 16 are also discussed herein. All uptake / delivery efficiencies are in Table 17 and are relative to the uptake / delivery efficiency of cFΦR4 (290-1F, 100%). SUV1 is a small unilamellar vesicle that mimics the neutral outer membrane of mammalian cells [45% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), 2% sphingomyelin (SM), and 15% cholesterol (CHO)]. SUV2 is a small unilamellar vesicle that mimics the negatively charged endosomal membrane of mammalian cells [50% PC, 20% PE, 10% phosphatidylinositol (PI), and 20% bis(monoacylglycerol) phosphate].

[0582] Fluorescence polarization measurements were performed on increasing concentrations of vesicles using FITC-labeled cyclic peptides. Experiments were performed at pH 7.4 and 5.5 (pH within late endosomes).

[0583] The overall delivery efficiency appears to be correlated with the binding affinity of the CPP to the endosomal membrane at pH 7.4, ie, tighter binding leads to higher delivery efficiency.

[0584] Table 16. Cyclic CPPs and their cellular uptake and membrane binding properties.

[0585]

[0586]

[0587] Φ = L-naphthylalanine; φ = D-naphthylalanine; f = D-phenylalanine; r = D-arginine; q = D-glutamine

[0588] Example 5

[0589] Cardiomyocytes are generally difficult to transfect with DNA and delivery of proteins therein by using previous CPPs has been unsuccessful. Thus, there is an unmet need for delivery of therapeutic proteins into cardiac tissue.

[0590] The disclosed cyclic CPPs are very effective in delivering proteins to cardiomyocytes. Fluorescein isothiocyanate (FITC)-labeled cyclic CPPs [c(FΦRRRRQ)-K(FITC)-NH2 and c(fΦRrRrQ)-K(FITC)-NH2] were synthesized and tested for internalization into mouse ventricular cardiomyocytes by treating cells with 5 μM FITC-labeled peptides for 3 hours. After washing away the extracellular peptides, the internalization of the CPPs was examined by fluorescent live cell confocal microscopy. Both peptides showed significant and mainly diffuse fluorescence throughout the cells, indicating that the CPPs were effectively internalized into cardiomyocytes (Figures 35a and 35b). Whether the cyclic CPPs are able to transport full-length proteins into cardiomyocytes was tested. Calmodulin (with engineered Thr5Cys) (a multifunctional calcium-binding messenger protein) was conjugated to c(FΦRRRRQ)-C-NH2 at a Cys residue near the N-terminus via a disulfide bond. Disulfide exchange reaction is highly specific, effective and reversible. In addition, when entering the cytosol of the cell, it is expected that disulfide bonds are reduced to release native proteins (Figure 35c). CPP-protein conjugates are chemically labeled with cyanine 3 on amino groups, which visualizes the internalization of calmodulin. Mouse ventricular cardiomyocytes were incubated with 6 μM CPP-calmodulin conjugates for 3 hours and examined by live cell confocal microscopy. Intracellular fluorescent signals are present in the entire cell volume and show sarcomere patterns (Figure 35d), indicating that internalized calmodulin is properly integrated into the cellular machinery. These data show that the disclosed cyclic CPP, such as c(FΦRRRRQ), is uniquely capable of delivering small molecules and proteins (possibly in its native form) to cardiomyocytes with high efficiency, thereby opening the door for future therapeutic applications.

[0591] Example 6

[0592] Pin1 is a phosphorylation-dependent peptidylprolyl cis / trans isomerase (PPIase). It contains an N-terminal WW domain and a C-terminal catalytic domain, both of which recognize specific phosphoserine (pSer) / phospho-threonine (pThr)-Pro motifs in its protein substrates. Through cis-trans isomerization of specific pSer / pThr-Pro bonds, Pin1 regulates the levels, activities, and intracellular localization of various phosphoproteins. For example, Pin1 controls the in vivo stability of cyclin D1 and cyclin E, and switches c-Jun, c-Fos, and NF-κB between their inactive, unstable and active stable forms. Isomerization of Pin1 also regulates the catalytic activity of many cell cycle signaling proteins such as the phosphatase CDC25C and the kinase Wee1. Finally, conformational changes in β-catenin and NF-κB catalyzed by Pin1 lead to subcellular translocation.

[0593] Given its key role in cell cycle regulation and increased expression levels and activity in human cancers, Pin1 has been proposed as a potential target for the development of anticancer drugs. Pin1 is also involved in neurodegenerative diseases such as Alzheimer's disease. Therefore, there is significant interest in developing specific inhibitors for Pin1. Small molecule inhibitors such as Juglone, PiB, dipentamethylenethiuram sulfide and halogenated phenylisothiazolone (TME-001) generally lack sufficient efficacy and / or specificity. Many effective peptidyl Pin1 inhibitors have been reported and are more selective than small molecule inhibitors. However, peptidyl inhibitors generally cannot permeate cell membranes, so their role as therapeutic agents or in vivo probes is limited. A cell-permeable bicyclic peptide-based inhibitor of Pin1, wherein one ring (Ring A) is characterized by a Pin1-binding phosphopeptide motif [D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acid and L-naphthylalanine, respectively], and the second ring (Ring B) contains the cell-penetrating peptide Phe-Nal-Arg-Arg-Arg-Arg shown in Fig.36 , peptide 1. Although the bicyclic peptide-based inhibitor was effective in cell-based assays (K D =72nM) and is active, and its D-pThr portion may be metabolically unstable due to hydrolysis by nonspecific phosphatases. The negative charge of the phosphate group may also hinder the cellular entry of the inhibitor. Here, non-phosphorylated bicyclic peptide-based inhibitors against Pin1 were prepared by screening peptide libraries and hit optimization. The resulting bicyclic peptide-based inhibitors are effective and selective for Pin1 in vitro, can permeate cells, and are metabolically stable in bioassays.

[0594] Although removal of the phosphoryl group of peptide 1 significantly reduced its potency against Pin1, the non-phosphorylated peptide ( Fig.36 , peptide 2) remains a relatively potent Pin1 inhibitor (K D =0.62 μM). The potency of peptide 2 can be further improved by optimizing the sequence flanking the D-Thr-Pip-Nal motif. 1 X 2 X 3 -Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-β-Ala-β-Ala-Pra-β-Ala-Hmb-β-Ala-β-Ala-Met-resin ( FIG. 35 , wherein Tm is trimesic acid, Dap is 2,3-diaminopropionic acid, β-Ala is β-alanine, Pra is L-propargylglycine, and Hmb is 4-hydroxymethylbenzoic acid) by randomizing the three N-terminal residues of peptide 2. 1 and X 2 The amino acid residues may be any one of the following 27 amino acid building blocks: 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp and Tyr], 5 non-proteinogenic α-L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (NIe), L-ornithine (Orn), L-phenylglycine (Phg) and L-Nal], 6 α-D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe and D-Val] and 4 N α -Methylated L-amino acids [LN α -Methylalanine (Mal), LN α -Methylleucine (Mle), LN α -methylphenylalanine (Mpa) and sarcosine (Sar)], while X 3 is Asp, Glu, D-Asp, D-Glu or D-Thr. The incorporation of these non-proteinogenic amino acids is expected to increase the structural diversity and proteolytic stability of the library peptides. The library has a theoretical diversity of 5×27×27 or 3645 different bicyclic peptides, most (if not all) of which are expected to be cell permeable. 5The library was synthesized on 100 μL of 100 μL beads / g, approximately 350 pmol peptide / bead). Peptide cyclization was achieved by forming three amide bonds between Tm and the N-terminal amine and side chain amines of two Dap residues.

[0595] Table 17. Hit sequences from peptide library screening a

[0596]

[0597] a Hits 1-3 were selected from the first screening round, while hits 4-7 were selected after the second screening round.

[0598] β-Ala provides a flexible linker, while Pra serves as a handle for labeling the bicyclic peptide beads with fluorescent probes via click chemistry. The ester linkage of Hmb enables selective release of the bicyclic peptide from the resin for solution binding analysis. Finally, the C-terminal Met allows release of the peptide from the resin by CNBr cleavage prior to MS analysis.

[0599] The library (100 mg resin) was screened for the S16A / Y23A mutant Pin1 with a defective WW domain. The mutant Pin1 was generated as a maltose binding protein (MBP) fusion at the N-terminus. During the first round of screening, Texas Red-labeled MBP-Pin1 was incubated with the peptide library and fluorescent beads were removed from the library under a microscope. Three positive beads had significantly higher fluorescence intensity than the remaining hits and were directly sequenced for peptides by partial Edman degradation mass spectrometry (PED-MS) (Table 17). The other 13 fluorescent beads were subjected to a second round of screening during which the bicyclic peptides on each bead were labeled with tetramethylrhodamine (TMR) azide at the Pra residue and released from the beads by treatment with NaOH solution.

[0600] Table 18. Sequences and Pin1 binding affinities of the peptides used

[0601]

[0602]

[0603]

[0604]

[0605] The released peptides were incubated with 5 μM MBP-Pin1 and the increase in fluorescence anisotropy (FA) was measured. For bicyclic peptides showing ≥50% increase in FA (relative to the no protein control), the corresponding beads (5 beads, which still contained the linear encoded peptide) were sequenced by PED-MS, resulting in 4 additional complete sequences (Table 1). All 7 hit sequences were in the X 3 The presence of a D-amino acid at position X is consistent with previous observations that Pin1 prefers D-pThr over pThr at this position. 1 There is a strong preference for hydrophobic residues, especially aromatic hydrophobic residues, but in X 2 There is no obvious selectivity in position.

[0606] Hit optimization. The six hit sequences (Hits 1 and 2 had the same sequence) were resynthesized with Lys added to their C-termini, labeled with fluorescein isothiocyanate (FITC), and tested for binding to Pin1 by FA (Table 18, peptides 3-8). All six peptides bound to Pin1 with moderate affinity (K D ~1 μM) bound to Pin1, but not to peptide 2 (K D =0.62 μM) did not improve. Peptides 3 and 4 were used for structure-activity relationship analysis and optimization. Expanding or contracting the size of the Pin1 binding loop (A loop) reduced the binding affinity (Table 18, peptides 9-16). Replacing the Ala residue of peptide 3 with amino acids containing side chains with different physicochemical properties, including Arg, Asp, Ser, Tyr and Val, also did not significantly improve the binding affinity (Table 18, peptides 17-21). On the other hand, modification of the D-Ala residue revealed that substitution of D-Phe at this position increased the Pin1 inhibitory activity by about 2-fold (K of peptide 22). D =0.48 μM).

[0607] Similar SAR studies were performed on peptide 4. As observed for peptide 3, modification of the Ala residue of peptide 4 (to Gly) had little effect (peptide 26), but replacement of the D-Ala residue with D-Phe increased the binding affinity for Pin1 by about 2-fold (Table 18, K for peptide 27). D =0.27 μM). 2 Modification of the Fpa residue at position (e.g., replacement with other halogenated phenylalanine analogs) all reduced inhibitor potency (peptides 28-32). 1Removal of the aromatic side chain at position 1 was detrimental to Pin1 binding (peptides 33 and 34). However, substitution with halogenated D-Phe analogs improved Pin1 binding activity (peptides 35-38). In particular, replacement of D-Phe with D-4-fluorophenylalanine (D-Fpa) resulted in the most potent Pin1 inhibitor of the series (K of peptide 37 was 2.34). D =0.12 μM)( Fig.36 Further attempts to modify the D-Thr residue or the CPP motif failed to improve Pin1 activity (Table 18, peptides 39-45).

[0608] Biological evaluation. To determine whether peptide 37 binds to the catalytic site of Pin1, its ability to compete with peptide 1 for binding to Pin1 was examined by FA analysis. Peptide 1 has been shown previously to bind to the Pin1 active site. As expected, peptide 37 inhibited the binding of peptide 1 to Pin1 with an IC 50 The value was 190 nM (Figure 37b). Next, the catalytic activity of Pin1 towards the peptide substrate Suc-Ala-Glu-Pro-Phe-pNA was monitored in the presence of increasing concentrations of peptide 37. Peptide 37 inhibited Pin1 activity in a concentration-dependent manner with an IC 50 The value was 170 nM (Figure 37c). These results indicate that peptide 37 binds at (or near) the active site of Pin1.

[0609] The selectivity of peptide 37 was evaluated by two different assays. First, peptide 37 was tested for binding to an arbitrarily selected panel of proteins including bovine serum albumin (BSA), protein tyrosine phosphatase 1B, SHP1 and SHP2, Grb2 SH2 domain, Ras, and tumor necrosis factor-α. Peptide 37 bound weakly to BSA (K D Peptide 37 was tested for its potential inhibition of Pin4, FKBP12, and cyclophilin A (three other common human peptidyl-prolyl cis-trans isomerases). Although Pin4 is structurally similar to Pin1 and partially overlaps with its function, peptide 37 only slightly inhibited Pin4 (about 15% at 5 μM inhibitor), with an estimated IC 50 The value was about 34 μM ( FIG. 37 c ). Peptide 37 had no effect on the catalytic activity of FKBP12 or cyclophilin A at concentrations up to 5 μM. These data indicate that peptide 37 is a highly specific inhibitor of Pin1.

[0610] The metabolic stability of peptide 37 was evaluated by incubating peptide 37 in human serum for different time periods and analyzing the reaction mixture by reverse phase HPLC. Pin1 inhibitor 1 containing pThr was used as a control. After 6 hours of incubation, 97% of peptide 37 remained intact, while about 50% of the bicyclic peptide 1 was degraded after 3 hours (Figure 37d). The loss of peptide 1 was accompanied by the simultaneous appearance of new peaks in HPLC. Mass spectrometry analysis of the new species identified it as a dephosphorylated product of peptide 1 (peptide 2). This result is consistent with our previous observations that structurally constrained bicyclic peptides are highly resistant to proteolytic degradation. The D-pThr portion remains susceptible to hydrolysis by nonspecific phosphatases in human serum.

[0611] The cellular uptake efficiency of peptide 37, peptide 1 and previously reported membrane-impermeable monocyclic Pin1 inhibitors (Table 18, peptide 46) was evaluated by incubating HeLa cells with FITC-labeled peptides (5 μM) for 2 hours and quantifying the total intracellular fluorescence by flow cytometry. As expected, untreated cells and cells treated with peptide 46 showed almost no cellular fluorescence, with mean fluorescence intensity (MFI) values ​​of 101 and 193, respectively (Figure 38a). In contrast, cells treated with peptides 1 and 37 obtained MFI values ​​of 2562 and 8792, respectively. Therefore, compared with peptide 1, peptide 37 is internalized by HeLa cells about 4 times more effectively. It is speculated that the negatively charged phosphate group of peptide 1 electrostatically interacts with the positively charged CPP motif and reduces the cellular uptake efficiency of the latter.

[0612] Inhibition of Pin1 activity has previously been shown to reduce cell proliferation. The effect of peptide 37 on HeLa cell growth was examined by using the MTT cell viability assay. The membrane-impermeable peptide 46 and the cell-permeable but inactive (Pin1 binding defective) bicyclic peptide (Table 18, peptide 47) were used as controls. Peptide 37 inhibited HeLa cell growth in a concentration-dependent manner with an IC of 50 The value was 1.0 μM (Figure 38b). As expected, neither peptide 46 nor 47 had any effect on cell growth. Time course studies also showed significant growth inhibition (>60%) after 3 days of treatment with 5 μM peptide 37 but not with peptides 46 or 47. Phosphorylated bicyclic peptide 1 had an IC of 1.8 μM under similar test conditions. 50 value.

[0613] Finally, to determine that Pin1 is the molecular target of peptide 37 in vivo, the intracellular protein levels of the established Pin1 substrate promyelocytic leukemia protein (PML) were examined by Western blot analysis. Pin1 negatively regulates PML levels in a phosphorylation-dependent manner, and it is expected that inhibition of Pin1 activity will stabilize PML and increase its intracellular levels. In fact, treatment of HeLa cells with peptide 37 (0.2-5 μM) resulted in a concentration-dependent increase in PML levels (Figure 38c, d). The effect was already significant at 0.2 μM inhibitor (PML levels increased 1.8 times) and reached a plateau at about 1 μM (3.3 times increase). Again, bicyclic peptide 47 had no effect under the same conditions, while peptide 1 (positive control, 5 μM) increased PML levels by 3.1 times.

[0614] By screening a peptide library followed by conventional medicinal chemistry approaches, the first potent, selective, metabolically stable and cell-permeable peptide-based inhibitor of human Pin 1 has been disclosed. Its high potency and selectivity should make it a useful chemical probe for exploring the cellular function of Pin 1.

[0615] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.Publications cited herein and the materials they cite are specifically incorporated by reference.

[0616] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. A pharmaceutical composition comprising a cyclic peptide of formula IIa, IIb or IIc: or a pharmaceutically acceptable salt thereof, in: AA 1 It is D-phenylalanine; AA 2 It is L-naphthylalanine; AA 3 It is L-arginine; AA 4 It is D-arginine; AA 5 It is L-arginine; AA 6 It is D-arginine; m and n are each 0; p is 1; and AA 9 is L-glutamine; or AA 1 Is D-phenylalanine; AA 2 Is L-naphthylalanine; AA 3 It is L-arginine; AA 4 It is D-arginine; AA 5 It is L-arginine; AA 6 It is D-arginine; m is 0; n and p are each 1; AA 8 is L-arginine; and AA 9 is L-glutamine; or AA 1 Is L-phenylalanine; AA 2 Is D-phenylalanine; AA 3 It is L-naphthylalanine; AA 4 It is L-arginine; AA 5 It is D-arginine; AA 6 It is L-arginine; m is 0; n and p are each 1; AA 8 is D-arginine; and AA 9 It is L-glutamine; The cargo moiety comprises a detectable moiety, a therapeutic moiety, a targeting moiety, or a combination thereof.

2. The pharmaceutical composition according to claim 1, comprising a cyclic peptide of formula IIa: or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1, comprising a cyclic peptide of formula IIb: or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 1, comprising a cyclic peptide of formula IIc: or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein AA 1 It is D-phenylalanine; AA 2 It is L-naphthylalanine; AA 3 It is L-arginine; AA 4 It is D-arginine; AA 5 It is L-arginine; AA 6 It is D-arginine; m and n are each 0; p is 1; and AA 9 It's L-glutamine.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein AA 1 It is D-phenylalanine; AA 2 It is L-naphthylalanine; AA 3 It is L-arginine; AA 4 It is D-arginine; AA 5 It is L-arginine; AA 6 It is D-arginine; m is 0; n and p are each 1; AA 8 is L-arginine; and AA 9 It's L-glutamine.

7. The pharmaceutical composition according to any one of claims 1 to 4, wherein AA 1 It is L-phenylalanine; AA 2 It is D-phenylalanine; AA 3 It is L-naphthylalanine; AA 4 It is L-arginine; AA 5 It is D-arginine; AA 6 It is L-arginine; m is 0; n and p are each 1; AA 8 is D-arginine; and AA 9 It's L-glutamine.

8. The pharmaceutical composition of any one of claims 1-4, wherein the cargo moiety comprises a detectable moiety.

9. The pharmaceutical composition of any one of claims 1-4, wherein the cargo moiety comprises a therapeutic moiety.

10. The pharmaceutical composition of claim 9, wherein the therapeutic moiety comprises a therapeutic protein.

11. The pharmaceutical composition of claim 10, wherein the therapeutic protein comprises an enzyme.

12. The pharmaceutical composition of claim 10, wherein the therapeutic protein comprises an antibody.

13. The pharmaceutical composition of any one of claims 1-4, wherein the cargo moiety comprises a targeting moiety.

14. A pharmaceutical composition according to any one of claims 1 to 4 for use as a medicament.

15. Use of the pharmaceutical composition according to any one of claims 1 to 14 in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

16. Use according to claim 15, wherein the disease or condition is a metabolic disorder or condition.

17. The use according to claim 15, wherein the disease or condition is cancer.

18. The use according to claim 15, wherein the disease or condition is an immune disorder or condition.

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