Modified peptides and related uses

Abstract

The present invention provides a drug that interacts with AKT and modulates AKT activity. [Solution] Modified peptides including the following: (i) Amino acid sequence (X)-GRT-(Y)-TLC-(Z), or (ii) an amino acid sequence (X)-GRT-(Y)-TLC-(Z) having at least 40% sequence identity with respect to the amino acid sequence, Here, X is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, or a combination thereof; and Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an amine group, or a combination thereof.

Description

[Technical Field]

[0001] Cross-references to related applications This disclosure claims priority and interest to U.S. Provisional Patent Application No. 62 / 857,293, “Modified Peptides and Related Methods of Use,” filed on 5 June 2019, which is incorporated herein by reference in its entirety for all purposes. background

[0002] 1. Field of Discovery. This disclosure generally relates to modified therapeutic polypeptides, their compositions, and their use in organisms that require them to treat and / or prevent diseases, such as cancer. This concerns the method of giving.

[0003] 2. Background Information. The AGC serine / threonine protein family of kinases consists of 63 evolutionarily related kinases, including PDK1, PKB / AKT, SGK, PKC, PRK / PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK, NDR, LATS, CRIK, MAST, GRK, Sgk494, YANK, Aurora, and PLK. Different AGC kinase families exhibit several aspects of inhibition and activation mechanisms. They share. The conformation of the catalytic domain of many AGC kinases is kinase domain It is regulated by the adjustment of the conformation of the regulatory site of the PIF pocket, which is the lobule of the in. The PIF pocket acts like an ON / OFF switch for AGC kinases with various regulatory modes, namely PDK1, PKB / AKT, LATS, and Aurora kinase. The molecular probe that stabilizes the PIF pocket in the procedure is an activator, and the damaged site The stabilizing compound is an allosteric inhibitor (Leroux et al. (2018) Semin Cancer Biol 48:1-17. Doi: 10.1016).

[0004] Phosphate-threonine kinase AKT (also known as protein kinase B) is involved in various proteins. It phosphorylates protein substrates, regulating many important physiological processes such as the cell cycle, glucose metabolism, cell growth and survival, angiogenesis, and protein synthesis (Brazil, et al. (2002) Cell 111:293-303). The stimulation of this catalytic activity is caused by phosphatidylinositol 3 kinase. This arises from the PtdIns(3,4,5)P-dependent recruitment of AKT from the cytoplasm to the membrane. Similarly, phosphorylation of two regulatory residues, Thr-308 and Ser-473, is also involved. PDK-1-catalyzed phosphorylation of Thr-308 is required for AKT activity, and this activity is enhanced approximately tenfold by Ser-473 phosphorylation (Alessi, et al. (1996) EMBO J. 15:6541-6551; Brazil, et al. (2002) supra).

[0005] Protein kinase A (PKA) is ubiquitously expressed in mammalian cells and regulates important cellular processes such as growth, development, memory, metabolism, gene expression, and lipolysis. PKA holoenzymes are inactive polymorphic enzymes. It exists as a compound and consists of two catalytic (PKAc) and regulatory (PKA RI&RII) subunits. The binding of cAMP promotes the dissociation and activation of the catalytic subunits. Each catalytic subunit consists of a small leaf and a large leaf, with the active site forming a slit between the two leaves. The smaller leaves provide the ATP binding site, while the larger leaves provide the catalytic residue and the docking surface for the peptide / protein substrate. The activation loop of the larger leaves contains Thr-197, a phosphorylation site essential for catalysis (Adams, et al. (1995) Biochemistry 34:2447-2454).

[0006] Deregulation of the AKT signaling pathway could lead to cancer, neurodegenerative and psychiatric brain disorders, and infectious diseases. It is known to be directly related to some of the most common and incurable human disorders (Blain and Massague (2002) Nat. Med. 8:1076-1078; Brazil, et al. (2004) Trends Biochem. Sci. 29:233-242; Chen, et al. (2003) Cell 113:457-468; Colin, et al. (200 5) Eur. J. Neurosci. 21:1478-1488; Emamian, et al. (2004) Nat. Genetics 36:131-137; Griffin, et al. (2005) J. Neurochem. 93:105-117; Liang, et al. (2002) Nat. Med. 8:1153-1160; Shin, et al. (2002) Nat. Med. 8:1145-1152; Viglietto, et al. (2002) Nat. Med. 8:1136-1144; Ji & Liu (2008) Recent Pat Biotechnol. (3):218-26) Increased AKT activity is associated with the pathology of several types of the most common human malignancies. It is well established that it is part of the process (Brazil, et al. (2004) supra), including breast cancer, prostate cancer, lung cancer, gastrointestinal tumors, pancreatic cancer, hepatocellular carcinoma, thyroid cancer, and malignancies of the central nervous system (such as glioblastoma and glioma). AKT function and Alzheimer's Alzheimer's disease (AD), Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), amyotrophic lateral sclerosis (ALS) It has also been reported to be associated with several neurodegenerative brain disorders, such as ALS (Griffin, et al. (2005) supra; Colin, et al. (2005) supra; Saudou, et al. (1998) Cell 95:55-66; Chen, et al. (2003) supra; Emamian, et al. (2003) Neuron 38:375-387; Kaspar, et al. (2003) Science 301:839-842). Furthermore, several studies have shown that activation of PI3K-AKT signaling is a strategy used by viruses to delay apoptosis and prolong viral replication in both acute and persistent infections. It may also be possible that the prevention of cell death promotes virus-induced carcinogenesis. The accumulated evidence suggests that PI3K or AKT The activity of this substance is important for the survival of several viruses, including HIV and other types of viruses. This suggests (Ji & Liu (2008) Recent Pat Biotechnol. (3):218-26; Chugh, et al. (2008) Retrovirology vol. 5 11. doi:10.1186 / 1742-4690-5-11).

[0007] Disruptions in the AKT signaling pathway are also involved in schizophrenia (Emamian, et al. (2004) supra). A genetic association between the AKT1 gene and schizophrenia has been confirmed in European (Schwab, et al. (2005) Biol. Psychiatry 58:446-450) and Japanese (Ikeda, et al. (2004) Biol. Psychiatry 56:698-700) populations. Furthermore, the PKA signaling pathway is a risk factor for schizophrenia. It has been shown to mediate the interaction between DISC1 and PDE4B, which are genetic factors known to be associated with high levels of [unclear] (Millar, et al. (2005) Science 310:1187-1191).

[0008] Considering the association of AKT with some of the most common and incurable human diseases, including cancer, infectious diseases, neurodegeneration and mental disorders, there is a need in the art to identify agents that interact with AKT and modulate its activity. The present disclosure meets this need in the art. Summary

[0009] There is currently described an active therapeutic peptide that can target the catalytic activity of some members of the kinase AGC family to modulate a variety of cellular functions including, but not limited to, cell proliferation, cell survival, cell death, etc.

[0010] In one aspect, the present disclosure provides a modified peptide comprising (i) the amino acid sequence (X)-GRT-(Y)-TLC-(Z), or (ii) an amino acid sequence having at least 40% sequence identity to the amino acid sequence (X)-GRT-(Y)-TLC-(Z). In these formulas, X is a natural amino acid, unnatural amino acid, chemical modification of a natural or unnatural amino acid, acetyl group, lipid group, or a combination thereof; Y is a natural amino <l acid, unnatural amino acid, chemical modification of a natural or unnatural amino acid, or a combination thereof; Z is a natural amino acid, unnatural amino acid, chemical modification of a natural or unnatural amino acid, amine group, or a combination thereof.

[0011] The present disclosure also provides a modified peptide having the sequence (X)-(seq1)-(Y)-(seq2)-(Z), or an amino acid sequence having at least 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity to the amino acid sequence (X)-(seq1)-(Y)-(seq2)-(Z). In these formulas, seq1 is GRT ​, KGRT, VKGRT, RVKGRT, KRVKGRT, (Orn)-RVKGRT, or AKRVKGRT; seq2 is TLC , TLCG, TLCGR, TLCGRPE, TLCGRPEY, or TLCGRPE-(4-Cl-Phe); X is a natural a A amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, natural or non A chemical modification of a natural amino acid, or a combination thereof; Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an amine group, or a combination thereof.

[0012] Methods for inhibiting the activity of at least one enzyme selected from the group consisting of AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA (Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7 / cyclin H / MNAT1, CDC42 BPB (MRCKB), and Abl are also provided; and methods for inhibiting cell proliferation are also provided, as well as methods for preventing or treating cancer, infectious diseases, or neurodegenerative diseases or disorders.

[0013] The aforementioned general areas of utility are given only as examples and are not intended to limit the scope of this disclosure and the accompanying claims. Additional objectives and advantages relating to the compositions, methods, and processes of this disclosure will be understood by those skilled in the art in light of the claims, description, and examples herein. For example, various aspects and embodiments of this disclosure can be used in numerous combinations, all of which are expressly contemplated herein. These additional advantage objects and embodiments are expressly included within the scope of this disclosure. Publications and other materials used herein to illustrate the background of this disclosure and, in particular, to provide additional details relating to the practice are incorporated by reference.

[0014] The accompanying illustrations incorporated herein and forming part thereof illustrate several embodiments of the present disclosure and, together with the description, help to illustrate the principles of the present disclosure. The illustrations are intended solely to illustrate one embodiment of the present disclosure and should not be construed as limiting the present disclosure. Further objects, features and advantages of the inventions of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying illustrations illustrating exemplary embodiments of the present disclosure: [Brief explanation of the drawing]

[0015] [Figure 1A-1B] Exemplary intracellular molecular targets of compounds: (1A) Representative immunoblots from cell-based assays by probing with antibodies that recognize PI3K-P110, or phospho-PDK1 (Ser-241), total AKT1, or phospho-P53 (Ser-46). Cell-based assays after treating U251 human glioblastoma cells with 5 μM to 40 μM of exemplary compounds at different vehicle concentrations or different time intervals of 30 minutes, 2 hours, or 24 hours. (1B) Phospho-AKT1 (Thr-308), p-CRAF (Ser-259), phospho-Aurora A (Thr-288), or total Aurora A.

[0016] [Figure 2]Apoptosis and survival of U251 glioblastoma cells at different time intervals after treatment with exemplary compounds: Equal numbers of U251 human glioblastoma cells (5 x 10⁵) were plated onto coverslips and treated with either a vehicle (upper panel) or an exemplary compound (lower panel) for 20 minutes, 1, 2, or 3 days. A TUNEL fluorescence assay (green) was performed to visualize apoptotic cells, and DAPI staining was used to visualize the nuclei of all cells (blue). Indirect immunofluorescence staining of cleaved caspase-3 (red) was performed as another marker of apoptosis. Cells treated with the vehicle grew to much higher confluence compared to cells treated with the exemplary compound (compared to the DAPI signals in the upper and lower panels). Confocal images (20X) show the three channels (first three columns) and the merged image (last column) separately at the same Z-step. The images show the time-dependent increase in the rate of apoptosis of cells after treatment. After 3 days of treatment, nearly 100% of cells showed apoptosis. The exemplary compounds exhibit apoptosis in both TUNEL (green) and cleavage caspase-3 (red).

[0017] [Figure 3A-3B]Quantitative analysis of cell density and apoptosis rate at different time intervals: Equal numbers of cells were plated onto coverslips and treated with exemplary compounds and vehicles at different time intervals. Cells were stained with DAPI and TUNEL and analyzed by confocal microscopy. The histogram bars in Figure 3A reflect the average number of total cells (DAPI-positive cells) remaining on the coverslips counted in five separate confocal fields. The histogram bars in Figure 3B reflect the average percentage of apoptotic cells calculated by dividing the number of TUNEL-positive cells by the total number of cells (DAPI-positive cells) remaining on the coverslips counted in five separate confocal fields. Cells treated with the vehicle consistently showed higher densities over time, reaching near-complete confluence at 72 hours (not shown), whereas cells treated with the exemplary compound consistently showed a significant decrease in cell density over time (3A) and a time-dependent increase in the number of apoptotic cells (3B), to the point where nearly 100% of the cells underwent apoptosis after 72 hours of treatment.

[0018] [Figure 4] IC-50 of exemplary compounds in human breast, lung, hematological, and skin cancer cells: Representative plates of metastatic human breast cancer cells (BT-549 and MDA-MB-468), human acute promyelocytic leukemia (HL-60), multiple myeloma cancer cell line (RPMI-8226), small cell lung cancer cell line (DMS-114), human melanoma cancer cell line (SK-ML-5) after 3 days of treatment with the three exemplary compounds, vehicle (PBS), or control of other kinase inhibitor compounds in clinical trials (MK-2206) or in clinical use (imatinib or Gleevec), serial dilutions from 50 μM to 0.7 μM. This experiment demonstrates superior efficacy and IC-50 in the nanomolar range of the exemplary compounds compared to existing classes of drugs in the latest stages of market or clinical trials.

[0019] [Figure 5A-5C] (5A) Inhibition of tumor growth and subsequent topical administration of exemplary compounds in animal models of brain tumors (GBM), (5B) liver cancer (HCC), and metastatic breast cancer: The percentage change in tumor size at different time intervals after intratumoral injection (days 0, 3, 7, 11, 14, 17, and 20) in animals treated with the vehicle (n=8) or exemplary compounds (n=8), i.e., the P-value, reflects the statistical difference between the mean tumor sizes of the two groups at the same time interval, obtained from Student's t-test or Mann-Whitney test. Detailed explanation

[0020] Next, exemplary embodiments will be referenced in detail, and examples thereof will be illustrated in this description. In this regard, exemplary embodiments of the present invention may take different forms and should not be construed as being limited to the description herein. Accordingly, exemplary embodiments will be simply described below by reference to the figures in order to illustrate the aspects of this description. Where used herein, the terms "and / or" refer to one of the relevant listed items. This includes all possible combinations of multiple elements. Expressions such as "at least one" refer to the elements. If it precedes a list, it modifies the entire list of elements, but not the individual elements of the list.

[0021] Terms such as 1st, 2nd, 3rd may be used herein to describe various elements, components, regions, layers, and / or sections, but these elements, components, regions Layers and / or sections should not be limited by these terms. These terms do not define one element, component, region, layer, or section as one element, component, region, region, layer, or section of another. Used solely to distinguish between elements, components, regions, layers, or sections. Therefore, the first element, component, region, layer, or section discussed below may be referred to as the second element, component, region, layer, or section without departing from the teaching of this embodiment.

[0022] The terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Where used herein, the singular forms "a" and "an" are used. , and "the" are intended to include plurals unless the context clearly indicates otherwise. They are doing it.

[0023] Furthermore, as used herein, “contains” and / or “contains,” or “contains.” The term "and / or "includes" means that the functions, areas, integers, steps, and operations described are included. Specifies the existence of an operation, element, and / or component, but not one or more other functions, regions, integers, steps, operations, elements, components, and / or their groupings. This does not preclude the existence or addition of a "P" (presumably a specific feature or element).

[0024] As used herein, “about” means within the range of acceptable deviations of a particular value (i.e., limits of the measuring system) determined by a person skilled in the art, taking into account the stated value and the errors associated with the measurement of the problem and the measurement of a particular quantity. For example, “about” means one or more standards This means within the deviation, or within ±30%, 20%, 10%, or 5% of the stated value.

[0025] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as they would be generally understood by those skilled in the art to which this disclosure belongs. It will be further understood that terms as defined in commonly used dictionaries should be interpreted as having the same meaning as their meanings in the context of the relevant art and this disclosure. Furthermore, unless expressly defined herein, terms should not be interpreted in an ideal or overly formal sense.

[0026] AKT has emerged as a focal point in many signaling pathways, including glucose metabolism, transcription, and It regulates multiple cellular processes such as potosis, cell proliferation, angiogenesis, and cell motility (Brazil, et al. (2002) supra). Many of the kinases of substrates involved in these processes and In addition to functioning as a substrate, it forms complexes with other proteins that are not substrates, and these other proteins regulate the activity and function of AKT (Brazil, et al. (2002) supra).

[0027] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to any molecule having at least two amino acids, amino acid analogs, or derivatives linked by peptide bonds or other covalent bonds.

[0028] Physical interactions between AKT and PKAc have been confirmed. While full-length PKAc was found to potently inhibit the catalytic activity of AKT, active AKT increased the catalytic activity of PKA through a mechanism that increased the phosphorylation level of PKAc at Thr-197. Unexpectedly, short PKAc fragments also increased the catalytic activity of PKA. These peptides may also modulate AKT. Some peptides were found to activate AKT, while others inhibited AKT activity. In particular, a PKAc fragment adjacent to Thr-197 of PKAc, referred to herein as ZaTa, was sufficient to potently inhibit AKT in vitro and in vivo. ZaTa penetrated cells, co-localized with AKT, inhibited and redistributed AKT within cells, and altered the expression pattern of PKAc. ZaTa also disrupted the AKT-PKAc complex in both in vitro and in vivo. Substantial changes were observed in the morphology of neurites and axons. Treatment of cultured cells with ZaTa also induced dose-dependent inhibition of cell proliferation. Furthermore, lowering PKAc protein levels increased AKT protein levels both in vitro and in vivo. Therefore, PKAc and its fragments are useful in modulating the AKT signaling pathway, which is involved in the regulation of glucose metabolism, transcription, apoptosis, cell proliferation, angiogenesis, and cell motility. Furthermore, it has been found to promote the prevention or treatment of cancer, infectious diseases, autoimmune diseases, neurodegenerative diseases, and mental disorders.

[0029] To identify proteins that directly interact with AKT, a co-immunoprecipitation assay was performed. AKT was purified from brain lysates. Co-immunoprecipitation proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by mass spectrometry. Using this approach, the catalytic subunit of PKA (PKAc) was identified as an AKT-interacting protein. In several independent co-immunoprecipitation experiments using two different antibodies that recognize different epitopes on the AKT sequence, one antibody recognized phosphorylated AKT at Ser-473, while the other antibody was produced against the plextrin homology (PH) domain of AKT and against PKAc. As determined by Western blot analysis using antibodies, PKAc is in complex with AKT. It was detected. Unexpectedly, when the sample was treated with cAMP, it formed a complex with AKT and became large A significant amount of PKAc was immunoprecipitated, and the level of unbound PKAc to regulatory subunits increased. These data indicate a physical interaction between endogenous PKAc and AKT, which occurs after PKA activation.

[0030] To demonstrate the co-localization of PKAc and AKT, we confocalized the intracellular localization of PKAc and AKT using a fluorescent antibody. Analysis was performed using a point microscope. Neuroblast cells Neuro-2a (N2a) expressing endogenous levels of AKT and PKAc were double-labeled with anti-AKT (PH domain) and anti-PKAc antibodies. The strongest signals at endogenous levels of both molecules in cultured N2a cells were detected along neurites and in the neurite extension zone. However, NG-108 neurons, a somatic cell hybrid of glioblastoma and neuroblastoma, showed a more diffuse pattern of co-localization in the cytoplasm and weaker signals in the neurites. These data confirmed the results of co-immunoprecipitation experiments and demonstrated the role of AKT and PKAc interaction in the growth and branching of neuronal processes. The co-localization of AKT and PKAc in non-neuronal cell lines derived from malignant cells was also determined. The analyzed cell lines were HTB-126 cells derived from invasive ductal carcinoma, and peripheral normal cells from invasive ductal carcinoma. HTB-125 cells derived from breast tissue, and CRL-2865 cells derived from pleural effusion metastases in patients with ductal carcinoma. These were cells. In these cells, endogenous AKT was found in both normal and malignant human mammary cells. It co-localized with endogenous PKAc in specific intracellular compartments. AKT was found in the nucleus (both HTB-125 and HTB-126). It appeared to co-localize with PKAc in microtubule-like structures adjacent to the cell membrane (HTB-125). These data indicate that the AKT-PKAc interaction is not specific to nerve cells and occurs in normal and malignant cell lines derived from human tissue.

[0031] To evaluate the importance of the AKT-PKAc interaction, kinase inhibitors were used. Highly selective and well-characterized PKA inhibitors and activators are well known in the art, however We then treated cultured neurons with H-89, a selective PKA inhibitor, and forskolin, a potent PKA activator, and analyzed AKT activity. Treatment with the PKA inhibitor caused a dose-dependent increase in AKT activity, while the PKA activator caused an increase at both the Thr-308 and Ser-473 sites. These had the opposite effect, as measured by the activation-dependent phosphorylation level of AAKT. The findings demonstrated that the level of AKT activity in cultured neurons is closely and inversely correlated with the level of PKA activity.

[0032] The observed effects of H-89 and forskolin on AKT activity in cultured cells are related to other signaling pathways. Since this could be interpreted as a result of regulatory interference in the pathway, we used purified active forms of AKT and PKAc to investigate the direct consequences of this interaction on kinase catalytic activity. In vitro analysis was performed. When full-length active PKAc was added to an AKT kinase assay containing active AKT as the kinase and Ser-9 GSK-3 glutathione (GST) fusion protein as the substrate, the kinetic activity of AKT dramatically decreased. This decrease in the catalytic activity of AKT by PKAc suggests that PKA inhibitors... It was inhibited when added to the reaction mixture. This observation indicated that the activity of PKA is required for its inhibitory effect on AKT. The reduction in catalytic activity of AKT by PKAc was observed in two mutants and AK This was also observed in the active form of T1, one with a deletion in the PH domain and the other with a Ser-473Asp mutation. Similar to wild-type AKT, the inhibitory effect of PKAc on the mutants was reversed in the presence of the PKA inhibitor peptide. This indicates that phosphorylation of the PH domain of AKT and at Ser-473 is not required for the inhibitory effect of PKAc on AKT. Furthermore, the phosphatase in the kinase reaction... Active PKAc purified from bovine tissue, or expressed in Sf9 cells, regardless of the presence or absence of a seroglycan inhibitor. The same inhibitory effect was observed in human PKAc.

[0033] The effect of AKT on the catalytic activity of PKA was further analyzed using an in vitro kinase assay involving PKAc as the kinase and DARPP32 as the substrate. PKA phosphorylates DARPP-32 at the Thr-34 site, converting it into a potent inhibitor of protein phosphatase-1 (Huang, et al. (1999) J. Biol. Chem. 274:7870-7878). The inhibitory role of PKAc on AKT catalytic activity and In contrast, the addition of active AKT to the PKA kinase assay increased the catalytic activity of PKAc. This was determined by measuring the phosphorylation level of DARPP-32 at Thr-34 using a phosphorylation-specific antibody against this site. Unexpectedly, the increase in PKAc catalytic activity was accompanied by an increase in the phosphorylation level of PKAc at Thr-197. This indicates proper biological function and It is likely a residue in the activation loop of PKAc, which is essential for cell motility. (Abel, et al. (2001) supra; Cheng, et al. (1998) Proc. Natl. Acad. Sci. USA 95:9849-9854). Autophosphorylation and phosphorylation by PDK-1 are possible mechanisms for this phosphorylation of Thr-197 in PKAc. It is described as a zoon (Moore, et al. (2002) J. Biol. Chem. 277:47878-47884) The in vitro data disclosed herein show that AKT phosphorylates Thr-197 of PKAc. Kinase reactions were performed in the same reaction tube in the presence of specific substrates for both PKAc and AKT. In this case, a similar opposing effect on catalytic activity was observed. In the control assay, neither phosphorylation of the Ser-9 GSK-3 GST fusion protein by PKAc nor phosphorylation of recombinant DARPP-32 at Thr-34 by AKT was observed.

[0034] Potent inhibitory peptides are readily available and widely used for PKAc and protein Unlike kinase C (PKC), inhibitors of AKT have generally been scarce until recently (Brazil, et al. (2004) supra). The use of PKA variants has been empirically shown to facilitate the structural design of more selective AKT inhibitors (Breitenlechner, et al. (2005) J. Med. Chem. 48:163-170). Furthermore, the optimal substrate motif of AKT has been modified for the design of AKT inhibitors (Obata, et al. (2000) J. Biol. Chem. 275:36108-36115). Since full-length PKAc protein inhibited the catalytic activity of AKT, peptide libraries based on human (GENBANK Accession No. NP_002721; SEQ ID NO:1) and bovine (GENBANK Accession No. CAA47627; SEQ ID NO:2) PKAc protein sequences were designed and synthesized. This library contained 96 duplicate peptides covering the full-length protein sequences of human and bovine PKAc from the N-terminus to the C-terminus (Table 1). The library was extensively screened to identify PKAc fragments that mediate the inhibitory effect of PKAc on AKT.

[0035] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0036] Unexpectedly, individual peptides in the library showed significant inhibitory effects on AKT. These peptides included peptides 49 (SEQ ID NO: 56), 53 (SEQ ID NO: 60), 62 (SEQ ID NO: 69), 63 (SEQ ID NO: 70), and 64 (SEQ ID NO: 71). Combinations of consecutive duplicate peptide fragments were also assayed for their effect on the catalytic activity of AKT. When peptides 25 to 36 are combined (i.e., sequence numbers 32-43), peptides 37-48 are combined. Wase (i.e., Sequence ID 44) - 55) Significant inhibitory effects were also observed when peptides 49-60 were combined (i.e., SEQ ID NOs: 56-67) and when peptides 61-72 were combined (i.e., SEQ ID NOs: 68-79).

[0037] Of particular interest regarding its inhibitory activity against AKT is the peptide Ala-Lys-Arg-Val-Lys-Gly-Arg-Thr-Trp-Thr-Leu-Cys-Gly-Thr-Pro-Glu-Tyr ( This was sequence number 60. This peptide, called ZaTa, enhances the in vitro catalytic activity of AKT. It was sufficient to inhibit the process. After adding ZaTa peptide to the AKT1 kinase assay, the phosphorylation level of the Ser-9 GSK-3 GST substrate decreased significantly. Bell isolated in vitro kinase assay products by SDS-PAGE and performed Western blot analysis using a phosphorylation-specific antibody that specifically recognizes Ser-9 phosphorylated GSK-3β. As determined by this, the AKT1 kinase assay was significantly reduced after the addition of the ZaTa peptide. Furthermore, the inhibitory effect of the ZaTa peptide was compared to the adjacent peptide (Gly-Phe-Ala-Lys-Arg-Val-Lys-Gly-Arg-Thr-Trp-Thr-Leu; SEQ ID NO: 59) and the 11 N-terminal amino acid residues of the ZaTa peptide. It was compared with peptides that overlapped and possessed a Thr-197 phosphorylation site. In this assay, the level of γ-32P incorporation into the Ser-21GSK-3β substrate peptide was used as a measure of the in vitro catalytic activity of AKT1. Adjacent overlapping peptides failed to inhibit AKT1, but the ZaTa peptide strongly inhibited the catalytic activity of AKT1 in vitro (IC). 50 ~0.1 μM; Figure 1). The ZaTa peptide itself is It was not a substrate of AKT, as determined by a control kinase reaction containing only the peptide and AKT. This is because phosphorylation at Thr-197 by AKT itself inhibits AKT by PKAc. It was shown that the amino acid sequence, biochemical properties, and / or structure adjacent to the Thr-197 site are not necessary, but play a role in inhibiting AKT. The natural inhibitor of AKT, namely PKAc, is The resulting ZaTa peptide is a strongly inhibited AKT, which is an independent series of kinases. In the case of Sei, similar to AKT, it did not show any inhibitory effect on the catalytic activity of PKAc, a member of the AGC family of kinases.

[0038] Similar to its in vitro inhibitory activity, the ZaTa peptide fragment also potently and efficiently inhibits AKT in the brain. We were able to inhibit it precisely. After stereotactic injection of ZaTa peptide, a decrease in the phosphorylation level of AKT substrates in the striatum of the cerebral hemisphere was observed compared to the other hemisphere injected with DMSO as a vehicle. These in vivo immunofluorescence results showed a significant decrease in the phosphorylation level of AKT substrates in vivo one hour after stereotactic injection of ZaTa peptide. This was also confirmed by the Western blot analysis shown. As a specific substrate, the phosphorylation level of GSK-3β at Ser-9 was also evaluated. After stereotactic injection of ZaTa peptide into the striatum of one hemisphere, a specific decrease in GSK-3β phosphorylation level at Ser-9 was observed compared to the other hemisphere injected with DMSO as a vehicle. Since no changes in the phosphorylation levels of GSK-3β at Tyr-216 or GSK-3α at Tyr-279 were observed, the decrease was specific to the AKT phosphorylation site of GSK-3β at Ser-9. Furthermore, no significant changes in AKT substrate phosphorylation in the frontal lobe or cerebellum were observed between the two hemispheres. The in vivo reduction in phosphorylation of AKT substrates was more pronounced at the injection site. These data not only confirmed the inhibitory effect of ZaTa peptide on AKT catalytic activity in vivo, but also demonstrated the efficient distribution and absorption of ZaTa peptide throughout brain tissue.

[0039] To test the specificity / selectivity of ZaTa as an inhibitor of AKT1 against other major kinases Therefore, a series of in vitro kinase assays were performed with AKT1 IC50 and 10-fold higher concentrations. The following panel of 32 kinases was first tested in vitro using the active form of each kinase and specific substrates: AKT2, AKT3, PKA, PKCα, PKCγ, PI3Kβ, PI3Kδ, PI3Kγ, SGK, PAK2, PAK3, SAPK2 / p38, Abl, CaMKII, CDK1 / cyclinB, CDK5 / p35, CK1, CK2, CSK, GSK3α, GSK3β, JNK1α1, MAPK1, p70S6K, PDGFRα, PDGFRβ, PDK1, PKG1α, TrkB, JAK2, JAK3, Syk. At AKT1 IC50 (0.1 μM), ZaTa did not show significant inhibition against any of the above kinases in vitro. However, at 10 times higher concentrations, ZaTa is found in AKT2 (63%), PI3Kδ (72%), p70S6K (64%), SGK (73%), PAK3 (83%), JAK3 (79%), TrkB (84%), and Abl (42%). This suppressed the kinases. To confirm the in vitro inhibitory effect on these kinases within cells, Cell-based assays were used to determine the effect of labeled ZaTa on the phosphorylation levels of well-known intracellular substrates of each kinase (Zipfel, et al. (2004) Curr. Biol. 14:1222-1231; Wang, et al. (2003) Arch. Biochem. Biophys. 410:7-15; King, et al. (1998) Nature 396:180-183; Rangone, et al. (2004) Eur. J. Neurosci. 19:273-279; Middlemas). , et al. (1994) J. Biol. Chem. 269:5458-5466; Huang, et al. (1999) J. Biol. Chem. 274:7870-7878). The results of this cell-based kinase assay confirmed that ZaTa can inhibit p70S6K in addition to AKT. In contrast, intracellular entry of labeled ZaTa inhibited PI3K, SGK It did not cause inhibition of PAK3, JAK3, TrkB, or Abl. These kinases may be inhibited by ZaTa at higher concentrations in vitro. These data suggest that ZaTa inhibits nanomolar We demonstrated a selective inhibitory effect on AKT1 at certain concentrations. However, at micromolar concentrations, ZaTa may also inhibit other selected kinases, particularly p70s6K in cell-based assays. Given that there are no known inhibitors for most of the kinases mentioned above, it is conceivable that ZaTa could be used at micromolar concentrations in in vitro studies to inhibit the activity of selected kinases.

[0040] To analyze in vivo selectivity, ZaTa was injected into one hemisphere and DMSO into the other hemisphere, as described above, and a series of Western blot analyses were performed using phosphorylation-specific antibodies that recognize either the phosphorylated substrate or the kinase inhibited at high concentrations by ZaTa in vitro. The results of this analysis confirmed potent in vivo inhibition of p70S6K by ZaTa and weaker in vivo inhibition of Abl. For other kinases assayed, the in vivo inhibitory effect was also examined. No such effect was observed. Given the high functional and structural homology between p70S6K and AKT, the c-inhibitory effect of ZaTa on p70S6K was considered. Furthermore, phosphorylation-specific antibodies that recognize the phosphorylation consensus sites of these kinases were used to phosphorylate the substrates of PKA, PKC, and CDK. The in vivo effect of ZaTa on levels was analyzed. In contrast to a consistent decrease in phosphorylation of AKT substrates, significant changes in phosphorylation levels of PKA, PKC, and CDKs substrates were observed upon in vivo injection of ZaTa. It was not observed later.

[0041] As striatum-specific substrates of PKA and CDK5, the phosphorylation levels of DARPP-32 were measured at Thr-34 (PKA site) or Thr-75 (CDK5 site) (Huang, et al. (1999) supra). ZaTa did not induce any significant changes in DARPP-32 phosphorylation at either of these sites. Therefore, compared to other major kinase families expressed in the brain (i.e., PKA, PKC, and CDK), the ZaTa peptide fragment selectively inhibited AKT in vivo.

[0042] Peptides can be highly effective inhibitors because they efficiently bind to and inhibit enzyme activity. However, intracellular delivery of peptides may limit their use. Some of these peptides, known as cell-permeable peptides (CPPs), are approved for use in site-directed drug delivery. Aside from the peptide mentioned above, inhibitory peptides may limit intracellular accumulation in in vivo enzyme studies. It has properties. CPP neuropeptides function as neurotransmitters in the central and peripheral nervous systems. The primary structure of the ZaTa peptide (i.e., it has a basic arm of several basic residues at the N terminus and a polar arm consisting of several residues with a free hydroxyl group at the C terminus) Based on the peptide's in vivo inhibitory effect in the brain, whether ZaTa peptide is a CPP (Continuously Activated Peptide) The determination was made that the ZaTa peptide's N-terminus was labeled with a red fluorescent dye because the C-terminus is important for its inhibitory activity. The efficiency of the labeling process and the purity of the labeled peptides were evaluated by mass spectrometry.

[0043] It was found that the ZaTa peptide penetrates cells and co-localizes with AKT, and that this is how AKT interacts with ZaTa This indicates that the peptide is an intracellular target. The cellular localization patterns of the ZaTa peptide differed from cell to cell: some cells showed strong nuclear signaling, some showed cytoplasmic staining by aggregates, and some showed bright signaling on the cell membrane. These different intracellular localization patterns of ZaTa were usually accompanied by the redistribution of AKT to the ZaTa site. In addition to the cellular redistribution of AKT upon ZaTa entry, the phosphorylation level of the AKT substrate in these cells was also observed. There was also a decrease in the prefrontal cortex. Similar results were obtained in vivo after stereotactic injection of fluorescent ZaTa into the prefrontal cortex. A specific decrease in the phosphorylation level of AKT substrates was observed in ZaTa-positive cells. These in vitro and in vivo observations suggest that ZaTa not only co-localizes with AKT in cells, but also its catalytic activity It was also shown to inhibit it.

[0044] When ZaTa enters cells, the expression pattern of PKAc differs depending on the localization of ZaTa. For example, cells showing strong nuclear signaling for ZaTa showed a significant decrease in PKAc immunoreactivity, while cells containing cytoplasmic aggregates of ZaTa generally showed elevated PKAc protein levels. These data suggest that appropriate intracellular AKT activity may influence PKAc expression. While I don't want to be bound by theory, the nuclear redistribution of AKT due to treatment with ZaTa suggests that PKAc expression is affected. It is thought that this caused repressive transcriptional changes. Alternatively, the redistribution of ZaTa within the cytoplasmic compartment may have a compensatory effect, namely an increase in PKAc activity, to compensate for the decrease in AKT activity. This may have caused the regulation.

[0045] The phenotypic consequences of disrupting the AKT-PKAc complex were also determined. ZaTa peptide was injected into the striatum of one hemisphere of the brain, and DMSO was administered as a medium to the other hemisphere of anesthetized adult C57BL / 6 mice. The substance was injected into the brain hemispheres. The brains were removed and dissected. Equal amounts of protein from each hemisphere were subjected to immunoprecipitation with anti-AKT antibody. Immunoprecipitation was performed from the right (vehicle-treated) and left (ZaTa-treated) striatum. The amount of precipitated AKT protein was comparable; however, the amount of PKAc in physical contact with AKT decreased dramatically after treatment with ZaTa. This indicated that ZaTa may disrupt the physical complex between AKT and PKAc in vivo. Western blot analysis was performed to compare PKAc protein levels in brain hemisphere lysates treated with ZaTa and vehicle. Similar amounts of PKAc were present in both hemispheres, but a clear increase in molecular weight was observed in the PKAc of the hemisphere treated with ZaTa. This suggests that treatment with ZaTa caused a change in the electrical mobility of PKAc, likely due to post-translational changes in the PKAc molecule.

[0046] As disclosed herein, N2a cells exhibited a neurite-specific pattern of AKT-PKAc interactions in their neuronal processes. Therefore, the stability of the AKT-PKAc complex and Phenotypic results were also analyzed using cultured neurons. N2a cells were used as a vehicle, ZaTa, or control peplo Cells were treated with cytoplasmic acid for 24 hours. After 24 hours of treatment, the culture medium was removed, and the same number of cells from each treatment group were either cultured for another 24 hours without treatment, or lysed and immunosuppressed with an antibody against AKT. The cells were subjected to immunoprecipitation. Cells cultured for 24 hours were also collected and subjected to immunoprecipitation. In parallel, the number of individual neurons with neurites was counted. Treatment with ZaTa involved physical contact with AKT. The amount of PKAc was reduced. This effect could be reversed by 24-hour incubation in the absence of ZaTa. At the same time, cells treated with ZaTa showed a significant decrease in the number of neurons with neurites (Figure 2). This was reversed by 24-hour incubation in the absence of ZaTa. The effect was reversible by incubation. These data not only confirmed in vivo observations showing ZaTa-induced disruption of the AKT-PKAc complex, but also demonstrated a correlation between neurite formation and the amount of PKAc physically in contact with AKT in cultured neurons. Furthermore, these data indicate that the effect of ZaTa is reversible. Live images of N2a neurons were taken from the vehicle or ZaTa Images were captured after treatment with (2 or 5 μM) of untreated N2a cells. It shows a normal pattern of neurite morphology in the region, and the treatment of N2a cells by ZaTa peptide The drug induced dramatic morphological changes in a dose-dependent manner. ZaTa-mediated changes included progressive loss of neurites, inhibition of new neurite formation, loss of cell motility, and the formation of large cell colonies.

[0047] To determine the phenotypic effect of disrupting the AKT-PKAc complex in an in vivo setting, the ZaTa peptide was used. The drug was injected stereotactically into the brains of mice, and the animals were perfused 18 hours after recovery from surgery. AKT is axonal. Because it is known to play a role in the state (Markus, et al. (2002) Neuron 35:65-76) Coronal sections of the striatum are markers for neurofilament-H (NF-H) as an axon-specific marker. Staining was performed. Changes in the staining pattern of striatal axonal filament bundles were observed during stereotactic injection of ZaTa peptide compared to other hemispheres injected with DMSO as a vehicle. To rule out the effects of tissue damage and to show that the striatal tissue structure was maintained postoperatively, sections were marked with nuclear markers. Co-labeling with (Draq5) was performed. NF-H and nuclear marker staining at the same Z-step showed similar histological structures in both the vehicle and ZaTa-treated hemispheres of the same coronal section. These data are consistent with co-localization observations disclosed herein and demonstrate the role of AKT in accelerating axonal growth and regeneration. (Markus, et al. (2002) supra; Namikawa, et al. (2000) J. See also Neurosci. 20:2875-2886). PKA is also known to play a role in the regeneration of growth cones on axons (Chierzi, et al. (2005) Eur. J. Neurosci. 21:2051-2062). Therefore, considering the data provided herein, the appropriate phase between AKT and PKAc It is believed that these interactions are involved in maintaining normal neuronal morphology.

[0048] AKT promotes G1 / S through several mechanisms, including the expression and intracellular localization of the CDK inhibitor p27Kip1. It influences networks that actively regulate cell cycle progression (Blain and Massague (2002) Nat. Med. 8:1076-1078; Liang, et al. (2002) supra; Shin, et al. (2002) supra; Viglietto, et al. (2002) supra). Based on these studies, the effect of ZaTa peptide on cell proliferation was evaluated. An MTT-based proliferation assay was used to assess the dose-dependent number of viable cells. A substantial decrease was observed. Western blot analysis showed a simultaneous decrease in AKT substrate phosphorylation levels in N2a cells after treatment with different doses of ZaTa peptide. Live images of N2a cells cultured in the presence of different concentrations of ZaTa peptide showed a decrease in mitotic levels. A clear decrease in the number of cells was observed. Therefore, the product of AKT in cell cycle progression Consistent with previous reports indicating a polarized role, the data disclosed herein demonstrate the inhibitory role of ZaTa peptides in the rate of cell proliferation.

[0049] Based on the importance of the C-terminal arm of ZaTa for its inhibitory effect on AKT, residues Thr-8 and Thr-10 (Equivalent to Thr-197 of full-length PKAc), the free hydroxyl groups at Thr-14 and Tyr-17 are this peptide We determined whether it is important for the biological activity of the cydorhizon. The ZaTa peptide variant was also Thr-8 These were synthesized by replacing the Thr-10 residue with Asp (ZaTaT8D and ZaTaT10D, respectively), an amino acid with a negatively charged side chain. ZaTa mutants with a hydrophilic, positively charged amino acid Arg at either the Thr-14 or Tyr-17 position (ZaTaT14R and ZaTaY17R, respectively) were also synthesized. ) was also synthesized. Replacing either Thr-8 or Thr-10 with Asp significantly reduced the inhibitory effect of ZaTa on cell proliferation, and replacing Thr-14 or Tyr-17 with Arg increased ZaTa activity. The increase was significant (Figure 3, histogram series 1). The inhibitory effect of ZaTa peptide on cell proliferation was largely reversible after removal of ZaTa treatment (Figure 3, histogram series 2). Wild-type ZaTa caused a significant decrease in viable cell count at a low dose of 2 μM, but no significant difference in viable cell count was observed 24 hours after removal of 10 μM wild-type ZaTa or the DMSO vehicle. These observations indicate that the biochemical properties of the amino acid side chains constituting the primary structure of the ZaTa peptide are important for the biological effects of this peptide. Like other peptides, ZaTa can switch between alpha and beta secondary structures. One structure is active. One conformation is favorable, while the other structure creates an inactive form. Therefore, mutations to Arg at Thr-14 or Thr-17 cause ZaTa's structure to become inactive. While it appeared to stabilize the conformation, changing Thr-8 or Thr-10 to Asp was advantageous for generating inactive conformations.

[0050] In some cell proliferation assays, such as MTT, a decrease in the number of viable cells is attributed to a decrease in the number of dividing cells. It does not distinguish whether this is due to cytotoxicity and death. Therefore, in addition to capturing live images, cells were stained with trypan blue at various time intervals after treatment with ZaTa and counted using a hemocytometer and light microscope. The same dose-dependent decrease in the number of viable cells was observed. However, wild-type ZaTa and ZaTa T8D , or ZaTa T10D After processing, 12-72 When counted at various points in time, no significant increase in the number of dead cells was observed, but ZaTa T14R Or ZaTa Y17 After treatment, a significant increase in the number of dead cells was observed only after 72 hours. This observation suggests that the inhibitory effect of wild-type ZaTa on AKT is reversible, but ZaTa T14R Or ZaTa Y17R The mutant induces apoptosis and cell death by causing irreversible inhibition of AKT. This demonstrated the potential for a permanent change to occur. Alternatively, the free hydroxyl group of Thr-14 or Tyr-17 could create an unstable / cleavable bond between ZaTa and AKT, while Arg-14 or Arg-17 could make this bond more stable and incleavable.

[0051] The interaction between PKAc and AKT at the transcriptional level was also evaluated by reducing PKAc alpha protein levels through RNA interference. The reduction in PKAc levels resulted in an increase in AKT1 protein levels in non-neuronal HeLa cells and neuronal NG-108 cells. Expression was also analyzed in PRKACA(PKAc alpha) knockout mice. Since homozygous knockout mice of this strain do not survive to adulthood, AKT1 protein levels were measured in heterozygous PKAc mice that express approximately 50% of the PKAc protein compared to wild-type mice. Protein extracts from the frontal cortex of heterozygous PKAc mice showed increased AKT1 protein levels. These data indicate that, in addition to physical interactions between AKT and PKA, an active transcriptional mechanism is involved that directly affects their activity levels and also regulates their protein levels.

[0052] It is now well established that AKT protects against apoptosis through phosphorylation and inhibition of apoptosis-promoting mediators such as BAD, FOXO family members, and IKK-β (Datta, et al. (1999) Genes Dev. 13:2905-2927). The effect of ZaTa on the protective function of AKT is... To demonstrate this, non-proliferating neurons in primary cortical cultures were subjected to the minimum AKT cell proliferation activity. The system was analyzed as a model system utilizing levels. Primary neurons were treated with either DMSO, 1 μM or 5 μM ZaTa, or a control peptide for 1, 3, 16, 24, 48, or 72 hours. The number of apoptotic cells was counted following the TUNEL assay. The results of this experiment were as follows: Compared to the control peptide, there was a significant dose-dependent increase in the number of TUNEL-positive cells after treatment with ZaTa for 72 hours. It showed a persistent increase. The increase in the number of apoptotic neurons after treatment with ZaTa was in AKT. This was consistent with strong intraneuronal inhibition.

[0053] To confirm the apoptosis-inducing effect of ZaTa in vivo, ZaTa was delivered to the brains of mice via the nasal cavity, a less invasive procedure compared to stereotactic surgery. Intranasal delivery of compounds to the brain is an efficient and effective method for local delivery of compounds without the need to cross the blood-brain barrier, which is a major obstacle to studying the effects of various inhibitors / activators in the CNS (Vyas, et al. (2005) Curr. Drug Deliv. 2:165-175; Hrafnkelsdottir, et al. (2005) Biol. Pharm. Bull. 28:1038-1042). Nasal delivery of labeled ZaTa to C57BL / 6 mice. Repeating intracavitary treatment for three days revealed apoptosis in the olfactory bulb, particularly in cells stained positively with ZaTa. The number of cells increased significantly. This was achieved by double-labeling brain sections with fluorescent TUNEL and nuclear markers. This was visualized by [method / analysis]. In contrast, no change in the number of apoptotic cells was observed in cells stained negatively for ZaTa or after treatment with the control peptide. Taken together, these data indicate that ZaTa can inhibit AKT-dependent function both in vitro and in vivo.

[0054] To improve the in vitro activity, in vivo efficacy, and stability of ZaTa, a series of modified peptides were developed. Tide is prepared. Therefore, this disclosure relates to diseases or This invention relates to compositions of ZaTa-modified peptides for use in methods of altering kinase activity in the treatment of a condition. The disclosed compositions included herein include pharmaceutical compositions comprising a ZaTa-modified peptide mixed with a pharmaceutically acceptable carrier. ZaTa and its variants alter AKT activity. Because it was found to inhibit and / or modify the activity of other kinases, certain practical applications The application form includes a pharmaceutical composition containing one or more ZaTa analogs.

[0055] The rational design of modified peptides is facilitated by the known crystal structure of activated AKT complexed with GSK-3 peptide and AMP-PNP (Yang, et al. (2002) Nat. Struct. Biol. 9:940-944). This structure reveals the binding of the GSK-3 peptide via the AKT activation loop. The short sequence of the ZaTa peptide surrounding Thr-197 located in the activation loop of PKAc (sequence number) It is said that (60) is sufficient to inhibit AKT as potently as the full-length PKAc protein. The observation was made during the interaction between the active conformations of two molecules, Thr-197 This suggests that the residues adjacent to the site are essential and sufficient for this inhibition. While we do not wish to be bound by theory, in the active conformation of full-length PKAc, the particular residues surrounding Thr-197 The specific sequence is thought to dock with the active site of AKT, thereby preventing efficient phosphorylation of Thr-308 and / or binding of the GKS-3 substrate peptide to the AKT activation loop, causing AKT to fail to phosphorylate GSK-3 at the Ser-9 site. See other elements of this interaction. In contrast to the inhibitory effect on active PKAc, AKT phosphorylates PKAc at Thr-197, increasing its catalytic activity. The data disclosed herein show that this phosphorylation is effective against AKT. This indicates that it is not necessary for the inhibitory effect of PKAc. However, it suggests that it is not necessary for the more active PKAc. In addition to supporting the sexual state, this is due to the subsequent inhibitory effect of full-length PKAc on AKT. This provides a conformational change that exposes the residues surrounding the site. Therefore, as a structural model, the PKAc / AKT interaction functions as an ON / OFF switch for the molecule that first phosphorylates Thr-197 of PKAc. This makes the conformation of PKAc more active, and the conformation of AKT becomes inactive upon binding to the activation loop. In the analysis of cAMP-inducible activation of PKA, the crystal structure of the catalytic and regulatory (RIα) subunit of PKA in the complex was determined (Kim, et al. (2005) Science 307:690-696). This analysis showed that the PKA inhibitor peptide of the RI subunit inhibits PKAc catalytic activity. This shows that it is sufficient.

[0056] A series of modifications were applied to the ZaTa peptide sequence to identify the shortest active fragment of the ZaTa peptide. Several fragments from the ZaTa peptide were synthesized and tested in vitro by MTT assay to find the shortest sequence capable of exhibiting anti-cell proliferation activity. Experiments revealed a ZaTa mutant with the short sequence Lys-Gly-Arg-Thr-(1-Nal)-Thr-Leu-Cys. It was found that this was sufficient to inhibit cell proliferation.

[0057] Furthermore, the ability of ZaTa peptides to form dimers of a single Cys residue, and ZaTa peptides The effect of this dimerization on the activity level and potency of the drug was investigated both in vitro and in vivo. The analysis was performed using the following method. For in vitro testing, the anti-cell proliferation activity of ZaTa monomers was evaluated using the MTT assay. The dimer was compared to the monomer of the peptide. This experiment showed that the dimer was more potent in inhibiting cell proliferation compared to the monomer of the peptide. In an in vivo assay, U251 The tumor growth rate of xenografts was compared among animals treated with the vehicle, ZaTa peptide dimer, or monomer. This animal experiment also confirmed that the dimer form has better activity compared to the monomer.

[0058] Furthermore, typical peptide modifications at different residues, such as peptide pegylation and lipidation, were tested and studied both in vitro and in vivo. This study focused on peptide lipidation. We have shown that both and / or pegylation enhance the anti-cell proliferation activity of the peptide both in vitro and in vivo.

[0059] Other modifications to the peptide, such as C-terminal acylation and N-terminal amidation, also improved the anti-cell proliferation activity of the ZaTa peptide. However, fluorescent molecules such as FITC were used to visualize the peptide's activity. Adding [this] appeared to decrease the anti-cell proliferation activity of the ZaTa peptide. Importantly, replacing hydrophilic residues of ZaTa, such as Thr-15 and Tyr-17, with halogenated amino acid derivatives such as 4-Cl-Tyr, 4-F-Tyr, 4-Cl-Phe, or 4-F-Phe improved ZaTa activity. Furthermore, changing native amino acids such as Lys to similar non-proteinogenic amino acids such as Orn improved ZaTa activity. It also improved.

[0060] A single substitution of a residue, or a compound with similar properties in terms of hydrophobicity and positive, negative, or neutral charge. Alterations to the ZaTa peptide sequence, such as the simultaneous substitution of two or three residues by anoacids, did not significantly alter its anti-cell proliferation peptide activity. This experiment reflects the fact that the peptide sequence can tolerate several mutations without blocking its anti-cell proliferation activity.

[0061] We investigated whether inhibition of AKT1 activity is necessary for ZaTa's anti-cell proliferation activity. Several variants of ZaTa were synthesized with different truncations and mutations and tested during in vitro kinase assays against AKT1. This study showed that there are indeed several variants of ZaTa that do not significantly inhibit AKT1 but can still inhibit cell proliferation. This experiment suggests that inhibition of AKT1 is one of the mechanisms of action of ZaTa, and that the anti-cell proliferation activity of ZaTa variants does not require inhibition of AKT1. They indicated that they would not do so.

[0062] Using in vitro kinase assays, the kinase inhibition profiles of ZaTa mutants were tested against approximately 115 different kinases involved in cell proliferation. In addition to AKT1, AKT2, p70S6K, and Abl, various variants of the ZaTa peptide are used. This demonstrated that it could inhibit several other kinases in the nanomolar range. More specifically, the kinases inhibited in the nanomolar range at different mutant concentrations were as follows: AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA (Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7 / cyclin H / MNAT1, CDC42 BPB (MRCKB)

[0063] Therefore, in one embodiment, the present disclosure includes modified peptides including: (i) Amino acid sequence (X)-GRT-(Y)-TLC-(Z), or (ii) An amino acid sequence having at least 40% sequence identity to the amino acid sequence (X)-GRT-(Y)-TLC-(Z), where X is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, or a combination thereof; or a combination thereof; Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an amine group, or a combination thereof.

[0064] In any aspect or embodiment described herein, X may include an acetyl group, a lauroyl group, or a palmitoyl group located at its terminus; Y may be 1-Nal, 2-Nal ; Z may include an amino group located at its terminus.

[0065] In any aspect or embodiment described herein, the amino acid sequence may be (X 1 )-KGRT-(Y)-TLC-(Z). Here, X1 is the same as X above.

[0066] In any aspect or embodiment described herein, the amino acid sequence may be (X 2 )-VKGRT-(Y)-TLC-(Z). Here, X2 is the same as X above.

[0067] In any aspect or embodiment described herein, the amino acid sequence may be (X 3 )-RVKGRT-(Y)-TLCGRPE-(Z1). Here, X3 is the same as X above, and here, Z1 is the same as Z above.

[0068] In any aspect or embodiment described herein, the amino acid sequence may be (X 4)-KRVKGRT-(Y)-TLCGRPE-(Z1) is possible. Here, X4 is the same as X above, and here, Z1 is the same as Z above.

[0069] In any embodiment or aspect described herein, the amino acid sequence is (X 5 )-RVKGRT-(Y)-TLCGRPE-(Z 1 ) is possible. Here, X4 is the same as X above, and here Z1 is This is the same as Z above.

[0070] In any embodiment or aspect described herein, the amino acid sequence is (X 7 )-V-(X 8 It can be )-GRT-(Y)-TLC-(Z), where X7 is the same as X above, and X8 is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, or a combination thereof.

[0071] In any embodiment or aspect described herein, the amino acid sequence is (X 9 )-KV-(X 8 It can be )-GRT-(Y)-TLC-(Z). Here, X9 is the same as X above.

[0072] In another aspect, this disclosure includes modified peptides having the following formula: (X)-(seq1)-(Y)-(seq2)-(Z) or an amino acid sequence having at least 40% sequence identity with the amino acid sequence (X)-(seq1)-(Y)-(seq2)-(Z) Here, seq1 is GRT, KGRT, VKGRT, RVKGRT, KRVKGRT, (Orn)-RVKGRT, or AKRVKGRT; seq2 is TLC, TLC, TLCGR, TLCGRPE, TLCGRPEY, or TLCGRPE-(4-Cl-Phe); X is a natural amino acid, a non-natural amino acid, a chemically modified natural or non-natural amino acid, or acetyl A group, a lipid group, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, a chemically modified natural or non-natural amino acid, or so These are combinations; Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an amine group, or a combination thereof.

[0073] In any embodiment or aspect described herein, X may include an acetyl group or lipid group located at its terminal end. The lipid group may be a C6-C20 lipid group, for example, a lauroyl group or It may be a palmitoyl group.

[0074] In any aspect or embodiment described herein, Y may be 1-Nal, and Z may be It may contain an amino group located at its terminal end.

[0075] In any aspect or embodiment described herein, the non-natural amino acid is ornithine, naphthylalanine, 4-chlorophenylalanine, or a combination thereof.

[0076] In another aspect, the disclosure encompasses dimers of the modified peptides described above. In any aspect or embodiment described herein, the dimer may be a homodimer or a heterodimer and may contain a disulfide bond.

[0077] According to this disclosure, exemplary modified peptides may include the following structures: Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2(Cys-Cys dimer) Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2 Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2(Cys-Cys dimer) Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2 Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2(Cys-Cys dimer) Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2 Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2 Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2(Cys-Cys dimer) Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2 Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH2(Cys-Cys dimer) Ac-KGRT-(1-Nal)-TLC-NH2 Ac-KGRT-(1-Nal)-TLC-NH2(Cys-Cys dimer) Ac-VKGRT-(1-Nal)-TLC-NH2 Ac-VKGRT-(1-Nal)-TLC-NH2(Cys-Cys dimer) Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH2 Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH2(Cys-Cys dimer) Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH2 Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH2(Cys-Cys dimer) Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH2 Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH2(Cys-Cys dimer) Ac-(Orn)-GRT-(1-Nal)-TLC-(4-Cl-Phe)-NH2 Ac-(Orn)-GRT-(1-Nal)-TLC--(4-Cl-Phe)-NH2(Cys-Cys dimer) Ac-(Orn)-GRT-(1-Nal)-TLC-NH2 Ac-(Orn)-GRT-(1-Nal)-TLC-NH2(Cys-Cys dimer) Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH2 Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH2(Cys-Cys dimer) Ac-KVKGRT-(1-Nal)-TLC-NH2 Ac-KVKGRT-(1-Nal)-TLC-NH2(Cys-Cys dimer) Ac-RVKGRT-(1-Nal)-TLC-NH2 Ac-RVKGRT-(1-Nal)-TLC-NH2(Cys-Cys dimer).

[0078] In the structure shown above, "lauroyl" is n-dodecanoyl, "palmitoyl" is n-hexadecanoyl, "Ac" is acetyl, "Orn" is ornithine, "1-Nal" is 1-naphthylalanine, "2-Nal" is 2-naphthylalanine, "4-Cl-Phe" is 4-chlorophenylalanine, and "Cys" is cysteine.

[0079] As used herein, “modified peptide” in this disclosure encompasses polypeptides that are recombinantly produced, purified from natural sources, or chemically synthesized. For ease of yield and purification, it is customary in the art to produce proteins and their fragments by recombinant protein methodologies. The method (i.e., cell-based) generally involves isolating a nucleic acid molecule encoding the protein or fragment of interest, incorporating the nucleic acid molecule into a recombinant expression vector in a form suitable for protein or fragment expression in a host cell, and expressing the protein. A form suitable for expression means that the recombinant expression vector contains one or more regulatory sequences operably ligated to the nucleic acid molecule encoding the protein or fragment of interest in a manner that enables transcription of the nucleic acid into mRNA and translation into mRNA protein. The regulatory sequence may include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Such regulatory sequences and vectors encoding them are known to those skilled in the art and are provided by Goeddel, Gene Expression Technology. Referenced in: Methods in Enzymology 185, Academic Press, San Francisco, California. Diego (1990). Vectors suitable for recombinant protein expression in mammals, yeast, or prokaryotes include those supplied by STRATAGENE®, INVITROGEN®, Pharmacia, etc. These vectors are commercially available from the source. Many of these vectors help purify heterologous polypeptides, i.e., signal sequences for secretion and / or the target protein or fragment. It encodes other polypeptides. Preferably, the heterologous polypeptide has a specific cleavage site for removing the heterologous polypeptide from the protein of interest. Other useful heterologous polypeptides that can be fused to the protein of interest either increase the expression or solubility of the fusion protein or assist in the purification of the fusion protein by acting as a ligand in affinity purification. Typical fusion expression vectors encode glutathione-S-transferase, maltose E-binding protein, or protein A. These include pGEX (Amersham Biosciences, Piscataway, New Jersey), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, NJ), which are fused to the target protein. Expression vector design is performed by transfecting It may depend on factors such as the selection of host cells to be used and / or the required level of expression. It should be understood that...

[0080] The introduction of recombinant expression vectors into host cells (e.g., those of eukaryotic or prokaryotic origin) can be carried out using any conventional technique for transforming cells. Appropriate methods for transforming host cells are described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2000)) and Other experimental manuals can be found. The number of host cells transformed with protein-coding nucleic acid molecules will depend, at least in part, on the type of recombinant expression vector used and the type of transformation technique used. Recombinant proteins or fragments can be expressed transiently or, more typically, stably, by incorporating the recombinant expression vector into the host cell genome or by episomal maintenance of the vector.

[0081] Once produced, the modified peptide can be recovered from the culture medium as a secreted polypeptide, or from the host cell lysate if it was expressed directly without a secretion signal. When the modified peptide is expressed in recombinant host cells of non-human origin, the modified peptide is substantially free of human-derived proteins or polypeptides. However, in order to obtain a preparation that is substantially homogeneous with respect to the modified peptide, it may be necessary to purify the modified peptide from recombinant cell proteins or polypeptides using conventional protein purification methods. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. Next, the membrane fraction and the soluble protein fraction are separated. The recombinant modified peptide can then be purified from the soluble protein fraction. Subsequently, the recombinant modified peptides are purified from the contaminant-soluble proteins and polypeptides using one of the following appropriate purification procedures: fractionation by immunoaffinity or ion exchange column; ethanol precipitation; reverse-phase HPLC; chromatography on cation exchange resins such as silica or DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using Sephadex® G-75, for example; and ligand affinity chromatography.

[0082] In addition to recombinant production, modified peptides can be produced by direct peptide synthesis using solid-phase technology (Merrifield RB (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis can be performed using manual or automated techniques. Automated synthesis can be achieved, for example, using the Applied Biosystems® 431A peptide synthesizer (Perkin Elmer, Boston, Massachusetts). When producing modified peptides, their various parts can be chemically synthesized separately and then combined using chemical methods to produce a full-length molecule.

[0083] Modified peptides, whether recombinantly produced or chemically synthesized, can be further functionalized for use. For example, modified peptides can enhance the activity of AKT and other kinases. Before use in inhibiting the target molecule, it can be phosphorylated, acetylated, methylated, or a combination thereof using well-known methods. Furthermore, PKAc and PKAc fragment-based therapeutics can be attached to modified peptide scaffolds.

[0084] In any aspect or embodiment described herein, the amino acid residues in the modified peptides of this disclosure are selected from any naturally occurring amino acids. In other embodiments, one or more or synthetic non-coding amino acids are used to replace one or more naturally occurring amino acid residues. Certain non-coding amino acids commonly encountered include: This includes, but is not limited to: peptide mimes or analogues; beta or gamma amino acids; D-enantiomers of genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); 2-aminoisobutyric acid (Aib); 6-aminohexanoic acid (Aha); 5-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methyl Soloisine (MeIle); Phenylglycine (Phg); Cyclohexylalanine (Cha); No Leleucine (Nle); homoleucine (hLle), homovaline (hVal); homoisolencin (hIle); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu ); 2,3-Diaminobutyric acid (Dab); N-methylvaline (MeVal); Homocysteine ​​(hCys); Homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro); etc. Additional non-coding amino acids are well known to those skilled in the art (e.g., Fasman (1989) CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Florida, Bo. See the various amino acids provided in Caraton, pages 3-70 and the references cited therein. Furthermore, the amino acids of the present invention may be in either the L configuration or the D configuration.

[0085] Modified peptides can reduce the expression or activity of AKT and / or other kinases, either as purified preparations or, in certain embodiments, formulated into pharmaceutical compositions containing an effective amount of the modified peptide or its dimer. Such pharmaceutical compositions can be prepared by methods and include carriers well known in the art. A generally accepted outline of such methods and components is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th publication, Lippincott Williams & Wilkins: Pencil Philadelphia, Rubenia, 2000. For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes, and even flavorings can be included in the pharmaceutical composition. For example, esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid can be added as preservatives. Furthermore, antioxidants and suspending agents can be used. Liposomes, as described in U.S. Patent No. 5,422,120, WO 95 / 13796, WO 91 / 14445, or EP 524,968B1, are also suitable carriers.

[0086] Depending on the intended use, the pharmaceutical compositions of this disclosure may be administered by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, intratumoral, or intramuscular injection), oral, or topical application (e.g., transdermally or via mucous membrane). A pharmaceutically acceptable formulation means a composition or formulation that enables the effective distribution of the peptide molecules of this disclosure at the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation using the peptide molecules of this disclosure include: PEG-bound nucleic acids, phospholipid-bound nucleic acids, and nucleic acids containing lipophilic moieties. Phosphothioates, P-glycoprotein inhibitors, can promote drug entry into various tissues. Drugs (such as Pluronic P85), e.g., CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); P-glycoprotein inhibitors (such as Pluronic P85) that can promote drug entry into various tissues (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Massachusetts; deliver drugs across the blood-brain barrier. Loaded nanoparticles, such as those made of polybutylcyanoacrylate, can alter the neuronal uptake mechanism (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies, including central nervous system delivery. This includes materials listed below: Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA, 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA, 96, 7053-7058. All of these references are incorporated herein by reference. The modified peptides of this disclosure were delivered to the brain via intranasal injection; the peptides were teto Dissolve in Laglycol (Sigma) to a final concentration of 0.5 mM, and inject 5 μl of the solution into each nostril. Ta.

[0087] In certain embodiments, the disclosure provides modified peptides that are cell-permeable peptides and rapidly distribute throughout human tissue. In certain embodiments, the peptides described herein are delivered locally to the site of a tumor. In exemplary embodiments, the modified peptides of the disclosure are injected directly into the site of a tumor, for example, through stereotactic surgery. However, one potential drawback of this method is that local injections are not generally formulated for sustained-release delivery.

[0088] Therefore, additional formulation / delivery devices are also envisioned that provide and / or adapt to the controlled and / or sustained release of the therapeutic agents of this disclosure. For example, modified Peptides are pharmaceutically acceptable and / or bioabsorbable carrier materials (i.e. (biologically inactive or biologically compatible), for example, conjugated into polymer matrices, biomolecular matrices, and / or other matrices. They can be captured (via covalent or non-covalent bonds), or simply captured. As used herein, “biologically inert or biologically compatible” means a material that does not produce a significant allergic or immunogenic response in a host. In one embodiment, the material consists of collagen. Other materials include proteins such as elastin, sugars and gels, and / Or a sol containing sugars, for example, hydroxypropyl cellulose (HPC), HPMC, metha This includes relates, etc. In exemplary embodiments, the material is an absorbable collagen sponge (ACS) or cross-linked collagen matrix, which is adapted to allow controlled and / or sustained release of peptides into tissue. Modified peptides The device or pre-molded / prefabricated mold is formed at the same time as, or after, the delivery device. It can be inserted into the TRICK material. In yet another embodiment, a modified peptide / biocompatible material (e.g., collagen) can be inserted into another device, which is also bioabsorbable and / or implantable, and the device is delivered to the tumor site for persistent local treatment. Enables delivery. The combination of modified peptides / biocompatible materials allows for delivery to another external device. It can also be inserted via [a specific method]. See McKay, B. Local Sustained Delivery of Recombinant Human Bone Morphogenetic Protein-2 (rhHBMP-2). 31st Annual International Conference of IEEE EMBS, September 2-6, 2009; Chan, BP, Feasibility study on the effects of photochemical crosslinking on collagen ultrastructure and controlled protein release. Acta Biomaterialia, 4:1627-36 (2008). These are incorporated herein by reference in their entirety.

[0089] The formulations may be administered orally, topically, parenterally, by inhalation or spray, or rectally in a dosage unit formulation comprising a conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, and vehicle. As used herein, the term parenteral includes transcutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques. Furthermore, formulations comprising the nucleic acid molecules and pharmaceutically acceptable carriers of the Disclosure are provided. One or more nucleic acid molecules of the Disclosure may be administered with one or more non-toxic, pharmaceutically acceptable carriers and / or diluents. may be present in association with and / or adjuvants, and other active ingredients as needed. The pharmaceutical compositions of this disclosure may be in forms suitable for oral use, for example, as tablets, lozenges, watery or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

[0090] Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more such sweeteners, flavorings, colorings or preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets contain an active ingredient mixed with non-toxic, pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients include, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate. Potential diluents may be active diluents; granulators and disintegrants, e.g., corn starch or alginate; binders, e.g., starch, gelatin or acacia; and lubricants, e.g., magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques. In some cases, such coatings may be prepared by known techniques to slow disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate may be used.

[0091] Oral formulations may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin, or olive oil.

[0092] The aqueous suspension comprises an active substance mixed with an excipient suitable for the preparation of the aqueous suspension. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents may be naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols. For example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with a partial ester derived from a fatty acid and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with a partial ester derived from a fatty acid and hexitol anhydride, such as polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives, for example It may contain ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[0093] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and flavorings can be added to provide a palatable oral formulation. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0094] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide active ingredients mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavorings, and colorants, may also be present.

[0095] The pharmaceutical compositions of this disclosure may also be in the form of oil-in-water emulsions. The oil phase may be vegetable oil or mineral oil or a mixture thereof. Suitable emulsifiers include naturally occurring gums, e.g., acacia gum or tragacanth gum; naturally occurring phosphatides, e.g., soybean, lecithin; and esters or partial esters derived from fatty acids and hexitol; anhydrides, e.g., sorbitan monooleate; and condensation products of the partial esters with ethylene oxide, e.g., polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavorings.

[0096] The syrup and elixir may contain sweeteners, such as glycerol, propylene glycol, sorbitol, glucose, or sucrose. It may also contain viscosity modifiers, preservatives, flavorings, and colorings. The pharmaceutical composition may be in the form of a sterile, injectable aqueous or oily suspension. This suspension may be formulated according to known techniques using the appropriate dispersants or wetting agents and suspending agents described above. The sterile, injectable preparation may also be a sterile, injectable solution or suspension in a non-toxic parenteral diluent or solvent, for example, as a solution of 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixative oils have traditionally been used as solvents or suspension media. For this purpose, any non-irritating fixative oil, including synthetic monoglycerides or diglycerides, can be used. Furthermore, fatty acids such as oleic acid have been used in the preparation of injectable preparations.

[0097] The peptide molecules of this disclosure can also be administered in the form of suppositories, for example, for rectal administration of the drug, or directly to the bladder itself via a catheter. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0098] The peptide molecules of this disclosure can be administered parenterally in sterile culture media. The drugs can be suspended or dissolved in the vehicle, depending on the vehicle and the concentration used. Advantageously, adjuvants such as local anesthetics, preservatives, and buffers can be dissolved in the vehicle.

[0099] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. The dosage unit is generally about 0.1 mg. It contains between approximately 1000 mg of active ingredients.

[0100] It is understood that the specific dose level for a particular patient or subject depends on a variety of factors, including: the activity of the specific compound used, age, weight, general health status, sex, diet, administration time, route of administration, excretion rate, drug combination, and the severity of the specific disease being treated.

[0101] The composition can also be added to animal feed or drinking water for administration to animals other than humans. It may be convenient to formulate the animal feed and drinking water compositions so that animals ingest therapeutically appropriate amounts of the composition along with their diet. It may also be convenient to present the composition as a premix for addition to supply water or drinking water.

[0102] The composition can also be administered to a target in combination with other therapeutic compounds to enhance the overall therapeutic effect. Using multiple compounds to treat an indication can increase beneficial effects while reducing the presence of side effects.

[0103] In certain embodiments, the disclosure includes host cells modified to harbor exogenous or heterologous nucleic acids, including nucleic acids encoding modified peptides.

[0104] The term "host cell" includes cells that can be used to carry heterologous nucleic acids, or cells that express peptides or proteins encoded by heterologous nucleic acids. Host cells may include genes not found in the cell's native (non-recombinant) form, genes found in the cell's native form that are modified and reintroduced into the cell by artificial means, or endogenous nucleic acids that are artificially modified without removing the nucleic acid from the cell. Host cells can be eukaryotes or prokaryotes. General growth conditions required for bacterial culture are described in BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, Vol. 1, NR Krieg, ed., Williams and This is described in texts such as Wilkins, Baltimore / London (1984). A “host cell” may also be one in which an endogenous gene or promoter, or both, has been modified to produce one or more polypeptide components of the complex of this disclosure.

[0105] Derivatives or variants of nucleic acids, proteins, or peptides of this disclosure include, but are not limited to,: molecules containing regions that are substantially homologous to the nucleic acids or proteins of this disclosure by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% identity (80-95% preferred identity) to a nucleic acid or amino acid sequence of the same size in various embodiments, or molecules when aligned to an aligned sequence, which is aligned by a computer homology program known in the art, or so A molecule in which the encoding nucleic acid can hybridize to the complement of the protein encoding sequence of the disclosed protein under stringent, moderately stringent, or low stringent conditions, or a molecule in which the encoding nucleic acid can hybridize to the complement of the protein encoding sequence of the disclosed protein under stringent, moderately stringent, or low stringent conditions. Ausubel, et al., CURRENT PROTOCOLS See, for example, IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993. Nucleic acid derivatives and modifications include gene substitution, site-directed mutation, deletion, insertion, recombination, repair, shuffling, endonuclease digestion, PCR, subcloning, and related processes. This includes things that can be obtained through related technologies.

[0106] Furthermore, one common technique would recognize that "conservative mutations" also include nucleic acid substitutions, deletions, or additions, where the nucleic acid change alters, adds, or deletes a single amino acid or a small number of amino acids in a coding sequence, resulting in the substitution of chemically similar amino acids. Amino acids that may serve as mutually conservative substitutions include: arginine (R), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E) ), asparagine (N), glutamine (Q); hydrophilic: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I); hydrophobic: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine ​​(C). Furthermore, the sequences that differ due to conservation variations are generally homologous.

[0107] Molecular biological techniques useful for implementing this disclosure, including mutagenesis, PCR, and cloning, are This is explained in the following papers: Berger and Kimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY, volume 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., MOLECULAR CLONING--A LABORATORY MANUAL (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.; Berger, Sambrook, and Ausubel, Mullis et al., U.S. Patent No. 4,683,202 (1987); PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al. eds), Academic Press, Inc., San Diego, Calif. (1990) (Innis);Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47 .

[0108] In yet another embodiment, the nucleic acids of this disclosure are expressed in mammalian cells using a mammalian expression vector. For expression systems suitable for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., old Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, as an example.

[0109] Polynucleotides can be DNA molecules, cDNA molecules, genomic DNA molecules, or RNA molecules. In the case of polynucleotides as DNA or RNA, T (thymidine) can also be U (uracil). A polynucleotide and a DNA or RNA molecule are complementary at a given position if a nucleotide at a specific position in the polynucleotide can form a Watson-Crick pairing with a nucleotide at the same position in an antiparallel DNA or RNA strand. A polynucleotide and a DNA or RNA molecule are substantially complementary if a sufficient number of corresponding positions within each molecule are occupied by nucleotides that can hybridize with one another to achieve a desired process.

[0110] Transformation of host cells by recombinant DNA is performed by conventional techniques well known to those skilled in the art. It can be carried out. "Transformation" refers to the process of introducing new DNA (i.e., exogenous DNA into the cell). This refers to permanent or transient genetic changes induced in cells following the uptake of DNA. ru.

[0111] In another embodiment, recombinant mammalian expression vectors can preferentially direct nucleic acid expression in a particular cell type (e.g., tissue-specific regulatory elements are used to express nucleic acids). Tissue-specific regulatory elements are known in the art. A non-limiting example of a suitable tissue-specific promoter is the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphocyte-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), especially T cell receptor promoters (Winoto and Baltimore, 1989. EMBO J. 8: 729-733), and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;;Queen and Baltimore, 1983. Cell 33: 741-748), New This includes rhinophyte-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Patent No. 264,166). This includes developmentally regulated promoters, such as the mouse homeotic promoter (Kessel and Gruss, 1990. Science 249: 374-379) and the α-fetoprotein promoter. This also includes Tarr (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

[0112] In any embodiment, the nucleic acid encoding the modified peptide of this disclosure may exist as follows: placed in a suitable expression vector and maintained episomalally. or multiple nuclei; one or more nucleic acids integrated into the genome of a host cell; components of a complex A modified version of an endogenous gene encoding; one or more nucleic acids combined with one or more regulatory nucleic acid sequences; or a combination thereof. The nucleic acids may optionally contain linker peptides or fusion protein components, such as His-Tag, FLAG-Tag, fluorescent proteins, GST, TAT, antibody moieties, signal peptides, etc., at their 5' end, 3' end, or anywhere within the ORF.

[0113] When the host is a prokaryote such as Escherichia coli, competent cells capable of DNA uptake can be prepared from cells collected after the exponential growth phase and then treated by the CaCl2 method according to procedures well known in the art, or by using MgCl2, RbCl, liposomes, or liposome-protein conjugates. Transformation can also be carried out after the formation of protoplasts in the host cells or by electroporation. These examples are not limiting to the disclosure; many techniques exist for transfecting host cells that are well known to those skilled in the art and are considered to be within the scope of the disclosure.

[0114] If the host is a eukaryote, such a method of transfection using DNA includes The calcium phosphate coprete is included, and conventional mechanical procedures such as microinjection, electroporation, insertion of liposome-encapsulated plasmids, or viral vectors, as well as other methods known in the art, can be used. Eukaryotic cells may be yeast cells (e.g., Saccharomyces cerevisiae) or include human cells. These cells may be mammalian cells. Stable expression is preferable for long-term, high-yield production of recombinant proteins.

[0115] As illustrated herein, the modified peptides of this disclosure are applied to inhibit the expression or activity of AKT and / or other kinases (as determined, for example, by phosphorylation of the Ser-21 GSK-3 substrate peptide), where kinase inhibition results in reduced cell proliferation and progressive, dose-dependent loss of existing neurites, as well as inhibition of new neurite formation. Thus, this disclosure encompasses not only the use of modified peptides to reduce cell proliferation, but also methods for preventing or treating cancerous or neurodegenerative or psychotic diseases or conditions.

[0116] The regulation of cell cycle progression via AKT is well-established in this field (see, for example, Brazil, et al. (2004) supra). AKT regulates the cell cycle by promoting G1 / S transition and the initiation of M phase (Collado, et al. (2000) J. Biol. Chem. 275:21960-21968; Datta, et al. (1999) Genes Dev. 13:2905-2927; Franke, et al. (1997) Cell 88:435-437). AKT also phosphorylates MDM2, causing it to translocate to the nucleus, where it promotes the degradation of p53 and p21. Cip1 It reduces mRNA transcription. In the nucleus, the FOXO transcription factor is p27 Kip1 Increase the transcription of However, this function is inhibited by AKT phosphorylation, and the FOXO protein remains in the cytoplasm. Clin-dependent kinase (CDK) inhibitor p21 Cip1 and p27 Kip1The protein is phosphorylated by AKT, accumulates in the cytoplasm, mitigates inhibition of CDK2 activity, and promotes G1 / S transition (Blain and Massague (2002) supra; Liang, et al. (2002) supra; Shin, et al. (2002) supra; Viglietto, et al. (2002) supra). AKT also drives the cell cycle to M phase by phosphorylating checkpoint proteins at FHA, the ring finger domain (CHFR), and Myt1 (Brazil, et al. (2004) supra; Okumura, et al. (2002) supra). AKT plays a crucial role in regulating multiple checkpoints during the cell cycle, and increased AKT activity is associated with breast cancer, Prostate cancer, lung cancer, gastrointestinal tumors, pancreatic cancer, hepatocellular carcinoma, thyroid cancer, CNS malignancies (thoracic cancer) Known to be involved in the most common human malignancies (such as glioblastoma and glioma), the modified peptides of this disclosure can be used, for example, to inhibit the proliferation of cancer cells in the prevention and treatment of cancer.

[0117] Glioblastoma is the most common primary central nervous system tumor in adults. Glioblastoma is characterized by high mitotic activity and marked vascular endothelial proliferation. Both of these mechanisms involve phosphorylation and It is strictly regulated by AKT via protein-protein interactions (Brazil, et al. (2002) supra). These features lead to a rapid growth rate, and most patients develop within one year of diagnosis. Death occurs within [number] years (Underwood (2004) General and systemic pathology. 4th Edition). The AKT signaling pathway is involved in tumor initiation and maintenance of glioblastoma and glioma (Lefranc, et al. (2005) J. Clin. Oncol. 23:2411-22; Kesari, et al. (2005) Curr. Neurol. Neurosci. Rep. 5:186-97), and targeting AKT may be effective in treating brain tumors. It is an effective strategy for this purpose (Kesari, et al. (2005) supra). Targeting AKT is an effective strategy for treating brain tumors (Carpentier (2005) Bull. Cancer 92:355-9). Monoclonal antibodies and small penetrating agents for local targeting of malignant gliomas. The effects of ptidohormones have been investigated (Merlo, et al. (2003) Acta Neurochir. Suppl. 88:83-91), and significant tumor uptake by small peptide hormone receptors has been observed.

[0118] The exemplary modified peptides disclosed herein are effectively absorbed and distributed throughout mouse brain tissue, and local administration of very small amounts of this peptide (only 1 μL of mM solution) specifically affects AKT. It was found to inhibit this process. The modified peptide also potently inhibits the proliferation of cancer cells derived from different malignant human cell lines. Three effects of the modified peptide, namely in brain tissue Considering the combination of distribution, inhibition of AKT and other peptides in vivo, and inhibition of cell proliferation, the modified peptide is found to affect human CNS tumors, and these three processes contribute to disease progression. It is useful in treating many other human malignancies that have been shown to play a significant role in poor prognosis. In the treatment of CNS tumors, the modified peptides of this disclosure are useful in highly invasive neurosurgical procedures. It has the advantage of being delivered directly to the tumor site using technology. Current treatments for CNS tumors are Typically, this involves either invasive neurosurgery or intensive radiation therapy, both of which carry the potential for serious postoperative complications.

[0119] Activation of the AKT pathway has been demonstrated to contribute to the pathogenesis of prostate cancer (Culig, et al. (2005) Endocr. Relat. Cancer 12:229-44), and inhibition of this signaling pathway is known to have therapeutic effects on human prostate adenocarcinoma (Wang, et al. (2004) Neuron 38:915-928). Therefore, targeting AKT with the modified peptides of this disclosure has been shown to have therapeutic effects on prostate cancer. It can also be used.

[0120] AKT has also been recorded as being involved in breast cancer. Peptide-based vaccines are common. Although it is used to target breast cancer (Disis, et al. (2004) Breast Dis. 20:3-11), the modified peptides of this disclosure can be used as first-line or adjuvant therapies in the treatment of breast cancer.

[0121] Methods for inhibiting cell proliferation generally involve the step of contacting cells (e.g., cancer cells) with an effective amount of a modified peptide (e.g., in a pharmaceutical composition). This reduces cell proliferation compared to cells that have not been contacted with the modified peptide. The reduction in cell proliferation is monitored. Means for doing so are disclosed herein.

[0122] In the context of cancer cell proliferation and the prevention or treatment of cancer, an effective dose is considered to be the amount that reduces or inhibits the proliferation of cancer cells to the extent that tumor development stops and / or tumor size decreases. This is done. Preferably, this drug reduces cancer cell proliferation or tumor size by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to other identical conditions where the modified peptide is absent. This can cause a 95% or 100% decrease.

[0123] As used herein, “effective dose” is used to refer to the amount of modified peptide required for the prevention, suppression of the onset of, or treatment (to some extent alleviate symptoms, preferably all symptoms) of a disease. The effective dose depends on the type of disease, the composition used, the route of administration, the type of animal being treated, the physical characteristics of the specific animal being considered for concomitant administration (age, weight, sex, etc.), and other factors recognized by the medical technician. Generally, the effective dose ranges from 0.001 mg / kg to 1000 mg / kg body weight / The daily dose is administered according to the potency. The invention of this disclosure includes a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of therapeutically and pharmaceutically acceptable excipients.

[0124] In one embodiment, the active compound is prepared using a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes that target infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These are methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811. It can be prepared according to the following.

[0125] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form. The dosage unit forms used herein are for the target being treated. This refers to physically distinct units suitable as a single dose; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the dosing unit forms in this disclosure are determined and directly depend on the inherent properties of the active compound, the specific therapeutic effect to be achieved, and the limitations inherent in the techniques for formulating such active compounds for the treatment of an individual.

[0126] The nucleic acid molecules of this disclosure can be inserted into a vector and used as gene therapy vectors. Gene therapy vectors can be delivered to a target, for example, by intravenous injection, local administration (see, for example, U.S. Patent No. 5,328,470), or stereotactic injection (see, for example, Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). A pharmaceutical preparation of a gene therapy vector may contain the gene therapy vector in an acceptable diluent, or it may contain a sustained-release matrix in which the gene delivery vehicle is embedded. Alternatively, if the complete gene delivery vector can be produced intact from recombinant cells, such as a retroviral vector, the pharmaceutical preparation may contain one or more cells that produce the gene delivery system. The pharmaceutical composition may be included in a container, pack, or dispenser along with administration instructions.

[0127] The toxicity and therapeutic effects of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, LD 50 (Lethal dose in 50% of the population) and ED 50 (For example, to determine the dose that is therapeutically effective in 50% of the population.) The dose ratio of toxic effect to therapeutic effect is the therapeutic index, LD 50 / ED 50 It can be expressed as a ratio of . It shows a large treatment index. Compounds are preferred. While compounds exhibiting toxic side effects can be used, care must be taken to design a delivery system that targets such compounds to the affected tissue site in order to minimize potential damage to uninfected cells and thereby reduce side effects. Data obtained from cell culture assays and animal experiments can be used in formulating a range of dosages for use in humans. Dosages of such compounds are preferably low- or no-toxic, ED. 50 The circulating concentration range includes [the specified compound]. The dose may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of this disclosure, the therapeutically effective dose can first be estimated from a cell culture assay. The IC determined in cell culture 50 Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration of the test compound that achieves half of the maximum inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high-performance liquid chromatography.

[0128] To the extent that this composition reduces or inhibits the proliferation of cancer cells, it is effective against breast cancer, prostate cancer, lung cancer, gastrointestinal tumors, pancreatic cancer, hepatocellular carcinoma, thyroid cancer, or CNS malignancies (such as glioblastoma and nerve cancer). Individuals who have or are at risk of developing cancer, such as glioma, would benefit from being treated with the modified peptides of this disclosure or compositions containing them. Individuals with cancer generally refer to patients diagnosed with cancer, but individuals at risk of cancer include those with a family history of cancer or one or more signs or symptoms associated with cancer (tumor, increased pain perception, weakness, etc.). Individuals may exhibit symptoms such as ( ). Such individuals, upon treatment with the compositions of this disclosure, are expected to show a reduction in cancer-related signs or symptoms, as well as a general improvement in quality of life and life expectancy. Not only are the compositions useful for the prevention or treatment of malignant tumors, but it is also believed that the compositions can be applied to the treatment of benign tumors, such as benign CNS tumors. Benign CNS tumors do not metastasize, but as a result of their high growth tendency in the skull and pressure on important CNS structures, they can cause serious complications and disorders. Therefore, treatment with the compositions of this invention would provide relief from such symptoms.

[0129] Given the enhanced cell-targeting activity of the modified peptides of this disclosure, certain embodiments may target cells. Alternatively, this includes the use of modified peptides as a component for the targeted delivery of therapeutic or contrast agents to tissues. Thus, instantaneously modified peptides can be operably linked to chemotherapeutic or therapeutic agents, for example, via covalent bonds, to increase cellular targeting and drug uptake compared to unbound agents. Alternatively, modified peptides can be attached to the surface of drug-loaded liposomes or nanoparticles to facilitate drug delivery to cells. Drugs that can target cells (e.g., cancer cells or neurons) using the modified peptides of this disclosure include cytotoxic agents such as: Taxol, Cytochalasin B, Gramicidin D, Ethidium Bromide, Emetine, Mitomycin, Etoposide, Teniposide, Vincristine, Vinblastine, Camptothecin (CPT), Colchkin, Doxorubicin, Daunorubicin, Mitoxantrone, Mitramycin Actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine tetracaine, lidocaine, propranolol, puromycin, therapeutic agents including antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracycline Antibiotics (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), anti-inflammatory agents, antimitotics (e.g., vincristine and Selective apoptotic agents such as vinblastine, APTOSYN(registered trademark)(Exisulind), PANZEM(trademark)(2-methoxyestradiol), and VELCADE(registered trademark)(Volte Zomib; proteasome inhibitors, cytotoxic agents, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, antibodies, kinase inhibitors, or other antitumor agents; radioisotopes, therapeutic nucleic acids or polypeptides, fluorescent markers, paramagnetic ions, contrast agents, metal chelators, toxins, hormones such as steroids; antimetabolites such as cytosine arabinoside, fluorouracil, methotrexate, or aminopterin; anthracyclines; mitomycin C; vinca alkaloids; demecolsin; etoposide; Mitramycin; or alkylating agents such as chlorambucil or melphalan, antitumor agents, chemotherapeutic agents such as nucleic acids, nucleotides, cytokines, antimetabolites, alkylating agents, antitumor agents, peptides or pseudopeptide chelating agents (e.g., linker chelating agents, glycyltyrosine (N,e-diethylenetriaminepentaacetic acid) hydrochloride lysine (GYK-DTPA-HCl), radioactive Compounds, diphtheria toxin (chain A), lysine toxin (chain A), adriamycin, chlorambucil, daunorubicin, or pokeweed antiviral proteins that enhance tumor-killing effects, nuclear magnetic spin resonance isotopes, metal ions, etc. However, as will be understood by those skilled in the art, this disclosure is not limited to a particular type or class of therapeutic agent or a particular disease being treated.

[0130] Methods for carrying out the binding of the above-mentioned drugs to peptides or pseudopeptides are well known or readily determinable, and include, for example, binding to amino acid side chains, functional groups, carbohydrates, lipids, and other small molecules. See, for example, Goldenberg, DM et al, New England J. Med., 298:1384-1388 (1978), Goldenberg, DM et al, JAMA, 250:630-635 (1983), Goldenberg. DM et al, Gastroenterol., 84:524-532 (1983), and Siccardi, AG et al, Cancer Res., 46:4817-4822 (1986). Furthermore, Epenetos, AA et al, Cancer, 55:984-987 (1985), Philben, VJ et al, Cancer, 57:571-576 (1986), Chiou, R. et al, Cancer Res., 45:6140-6146 (1985), and Hwang, KM et al, J. Natl. Cancer Inst., 76:849-855 (1986): these are all specifically incorporated herein by reference.

[0131] Examples of markers that can be conjugated to antibodies are well known to those skilled in the art and include substances that can be detected by nuclear magnetic resonance imaging: namely, nuclear magnetic spin resonance isotopes. , and radioactive materials. A preferred example of a nuclear magnetic spin resonance isotope is gadolinium (Gd). A suitable example of a radioactive marker is I 125 , I 131, I 123 In 111 In 113 , Ga 67 , Ga 68 , Ru 97 , Ru 103 Hg 197 Hg 203 , Tc 99 This includes the detection of radioactive markers, as described in the references above. As described, this is done using devices such as gun macination cameras. Nuclear magnetic imaging devices can be used to detect nuclear magnetic spin resonance isotope markers.

[0132] Generally, the modified peptides of this disclosure are amphiphilic with a net positive charge. Generally, amphiphilic structures play an important role in mediating interactions between peptides and proteins and membranes (Sharadadevi, et al. (2005) Proteins 59:791-801). Primary amphiphilic cell-permeable peptides have been used for drug delivery because they have been used for the efficient intracellular delivery of large hydrophilic molecules such as oligonucleotides and proteins (Plenat, et al. (2005) Biophys. J. 89:4300-4309). It has been shown that the generation of Arg or Lys is strongly preferred in amphiphilic helices (Sharadadevi, et al. (2005) supra). There is also a relationship between net charge and mean hydrophobic moment, which is a determinant of membrane-finding properties. Net positive charge appears to favor a higher hydrophobic moment than net negative charge (Sharadadevi, et al. (2005) supra). Similar to known cell-permeable peptides, the amphiphilic structure of the modified peptide facilitates penetration into cells and targets AKT, PKAc, and other kinases within the cell. and exerts its biological activity. Mutations to Arg at Thr-14 or Tyr-17 result in higher net activity. This generates a positive charge, thereby increasing the average hydrophobic moment of the modified peptide and enhancing its effect. On the other hand, mutating Thr-8 or 10 to a negatively charged Asp results in a reduced positive net charge and decreased biological activity. Therefore, the modified peptides of this disclosure are useful not only as anticancer agents but also for the targeted delivery of additional therapeutic agents to cells.

[0133] Several other peptide modifications in the ZaTa sequence improved its activity. Examples of such modifications include replacing one, two, or three amino acids, as well as native amino acids. This involves replacing the amino acids with abnormal ones, for example, changing Try to Nal, Lys to Orn, or replacing native hydrophilic residues such as Thr or Tyr with halogenated amino acid derivatives such as 4-ClTyr, 4-FTyr, 4-Cl-Phe, or 4-F-Phe.

[0134] Similar to cancer, increased AKT activity has been associated with different types of Alzheimer's disease. It is also known that (Blain and Massague, (2002) supra; Griffin, et al. (2005) supra; Liang, et al. (2002) supra; Shin, et al. (2002) supra; Viglietto, et al. (2002) supra), and decreased AKT activity is associated with schizophrenia (Emamian, et al. (2004)). supra). Moreover, the PKA signaling pathway has a novel role for in schizophrenia as well (Millar, et al. (2005) supra).

[0135] Regarding neuronal synaptic activity and neurodegeneration, ion channels are a new class of PKB / Akt substrates and It has been identified as such, and synaptic plasticity has been identified as a biological process regulated by this kinase. In particular, the β2 subunit of the type A γ-aminobutyric acid (GABAAA) receptor It is an AKT substrate in vitro and in vivo (Wang, et al. (2002) Neuron 38:915-928). This protein is a member of ligand-gated chloride ion channels that mediate synaptic transmission at most inhibitory synapses in the mammalian brain. Drugs such as benzodiazepines and barbiturates act on ABAAA receptors to mediate antipsychotic effects. AKT-mediated phosphorylation of Ser-410 increases the number of GABAAA receptors on the plasma membrane surface, thereby increasing the effectiveness of receptor-mediated inhibition at GABAergic synapses (Brazil, et al. (2004)). (super).

[0136] Human studies provide evidence of increased AKT activation and hyperphosphorylation of key AKT substrates in the brains of Alzheimer's disease (AD) patients (Griffin, et al. (2005) supra). Different distributions of AKT and phosphoAKT were observed in AD temporal cortical neurons compared to control neurons, which is due to particle This leads to increased AKT activation (phosphorylated AKT / total AKT ratio) in AD, accompanied by increased levels of active phosphorylated AKT in the fraction and a significant decrease in AKT levels in the AD cytosolic fraction. Furthermore, a significant increase in phosphorylation levels of total AKT substrates, including decreased levels of GSK3β (Ser-9), tau (Ser-214), mTOR (Ser-2448), and AKT target p27kip1, has been reported in the AD temporal cortex compared to controls. In addition, PTEN (staining No. 10), a major negative regulator of AKT, is also involved. Significant loss and altered distribution of phosphatases and tensin homologs (deleted on the chromosome) are observed in AD neurons. Loss of phosphorylated AKT and PTEN in neurons is indicative of AD. Observed in end-stage hippocampal CA1 (Griffin, et al. (2005) supra). AD cases and corresponding The enzymatic activity of AKT in the midtemporal and midfrontal lobes from the control group has also been analyzed (Rickle, et al. (2004) Neuroreport 15:955-959). The results of this analysis showed that AKT (immunoprecipitation AKT) The activity of GSK-3α / β fusion protein phosphorylation was significantly increased in the temporal cortex soluble fraction from AD compared to non-disease controls and positive disease controls with other neurodegenerative diseases. Furthermore, AKT activity in the temporal cortex soluble fraction was compared with the Blaak stage classification of neurofibrillary tangles. There was a positive correlation. Strong phosphorylated AKT immunoreactivity was associated with degenerated AD pyramidal neurons. This was demonstrated in reactive astrocytes. PKAc fragments strongly phosphorylate the AKT substrate in the brain. Considering that it can be reduced, inhibition of AKT is the same as the changes observed in humans with AD. This could reverse the trend and potentially bring therapeutic benefits to the treatment of Alzheimer's disease.

[0137] Many hereditary neurodegenerative diseases produce long polyglutamine (polyQ) sequences in proteins. This is caused by the expansion of CAG repeats, and their length is directly correlated with the severity of the disease. (Emamian, et al. (2003) Neuron 38:375-387; Chen, et al (2003) Cell 113:457-68). AKT substrates mediating the pathophysiology of spinocerebellar ataxia type 1 (SCA1) and Huntington's disease have been identified (Humbert, et al. (2002) Dev. Cell 2:831-837; Emamian, et al. (2003)). supra; Chen, et al. (2003) supra). The toxicity of the mutant protein in vivo is Ser-776 It is directly mediated by phosphorylation (Emamian, et al. (2003) supra). Replacing Ser-776 with Ala completely eliminates the pathogenesis in vivo, even in the presence of long polyglutamine sequences. It can be avoided. Therefore, polyglutamine expansion is necessary for the onset of the disease, but not sufficient on its own. Based on this analysis, Ser-776 was identified as the site of AKT phosphorylation. This is a molecular event essential for the interaction between 14-3-3 and polyQ-extended attaxin 1 (Chen, et al. (2003) supra), where binding to 14-3-3 triggers the formation of attaxin 1 inclusion bodies, which mediates its neurotoxicity. In this regard, PKB / Akt-mediated phosphorylation of mutant attaxin 1 in SCA1 is related to 14-3-3 binding, the stepwise accumulation of this protein, and This can lead to neurodegeneration as a result.

[0138] Similar to SCA1, Huntington's disease is characterized by an expansion of the polyQ repeat in the huntingtin protein. It is characterized by the aggregation of mutant proteins in the nucleus and selective apoptosis of striatal neurons in the brain (Saudou, et al. (1998) Cell 95:55-66). However, this is in contrast to its role in SCA1. Specifically, AKT evaluates the survival of striatal neurons lost during degeneration seen in Huntington's disease. Extreme regulation. Insulin-like growth factor 1 (IGF-1) therapy and AKT activation of striatal neurons. Both inhibit mutant Huntington's protein-mediated cell death and nuclear inclusion formation (Humbert, et al. (2002) supra). PKB / Akt-mediated mutant Huntington's protein on Ser-421 Phosphorylation of Huntington's protein is required for IGF-1-mediated nuclear inclusion body formation and inhibition of cell death, and a decrease in AKT activity may accelerate the progression of Huntington's disease. In this regard, analysis of AKT protein in brain samples from individuals affected by Huntington's disease revealed full-length AKT (60 kDa) and the cleavage of the full-length kinase via caspase-3, which is predicted to be produced. This reveals the existence of both shorter forms (49 kDa) (Humbert, et al. (2002) supra).

[0139] AKT signaling in neurons with amyotrophic lateral sclerosis (ALS) has also been determined (Kaspar, et al. (2003) supra). IGF-1 stimulates PKB / Akt activity in the spinal cord, and in this setting, motor By increasing neuronal survival, the lifespan of SOD1 mice was extended, and IGF-1 administration was This suggests it may be beneficial in the treatment of amyotrophic lateral sclerosis (Kaspar, et al.). (2003) supra).

[0140] Direct evidence has been provided regarding the role of AKT in accelerating axonal growth and axonal regeneration. It is present ((Markus, et al. (2002) supra; Namikawa, et al. (2000) Nat. Cell Biol. 4:111-116). Furthermore, PKA has been shown to play a role in axonal pathway exploration of olfactory neurons in zebrafish (Yoshida, et al. (2002) J. Neurosci. 22:4964-4972)), and in the ability of axons to regenerate growth cones (Chierzi, et al. (2005) supra). Since endogenous PKAc colocalizes with AKT along the length of neurites in N2a neurons and in the neurite extension zone, and we have demonstrated that treatment with the modified peptides of this disclosure results in progressive, dose-dependent loss of existing neurites and inhibition of new neurite formation, methods for modulating neurodegenerative and psychotic diseases and conditions are also encompassed by this disclosure. In particular, based on the role of AKT in the pathogenesis of SCA1, Huntington's disease, ALS, and AD, the modified peptides and their compositions may be useful in the prevention and treatment of these neurodegenerative diseases.

[0141] In another aspect, the Disclosure also provides a method for preventing or treating an immunodeficiency disorder, such as AIDS, using a modified peptide (e.g., in a pharmaceutical composition). As prophylactic or therapeutic, an effective amount of the Composition is administered to a patient (who has an immunodeficiency disorder, such as AIDS (e.g., exhibits signs or symptoms of the disease) or is at risk of having it (e.g., is genetically predisposed) in order to prevent (i.e., inhibit or delay the onset or development of the disease or disorder) or to treat (i.e., improve its signs or symptoms). The immunodeficiency diseases or conditions included in this disclosure include, but are not limited to, AIDS, leukemia, lymphoma, viral diseases, e.g., hepatitis, multiple myeloma, ataxia telangiectasia, Chediak-Higashi syndrome, combined immunodeficiency, complement deficiency, DiGeorge syndrome, hypogammaglobulinemia, Job's syndrome, leukocyte adhesion disorder, panhypogammaglobulinemia, Bruton's tyrosine kinase, congenital agammaglobulinemia, and IgA deficiency Selective deficiency, Wiscott-Aldrich syndrome.

[0142] In another aspect, the Disclosure also provides a method for preventing or treating neurodegenerative diseases or disorders using modified peptides (e.g., in pharmaceutical compositions). Whether prophylactic or therapeutic, an effective amount of the Composition prevents (i.e., treats) neurodegenerative diseases or disorders. It is administered to patients who have (e.g., exhibit signs or symptoms of) or are at risk (e.g., have a genetic predisposition) of a neurodegenerative disease or neurological disorder (or to inhibit or delay the onset of the disorder) or to treat (i.e., improve its signs or symptoms). Neurodegenerative diseases or conditions included in this disclosure include, but are not limited to, SCA1, Huntington's disease, ALS, and AD.

[0143] Large-scale gene therapy clinical trials for the treatment of Parkinson's disease are known (Howard (2003) Nat. Biotechnol. 21(10):1117-8). In these trials, genes are cloned into recombinant expression vectors known to be deregulated in the disease and delivered locally to the diseased area of ​​the brain. Therefore, these gene therapy approaches in clinical trials deliver DNA molecules encoding the PKAc protein or fragment to the site of pathology. The aim is to enable the use of the same settings. This approach can be used to overexpress PKAc protein or fragments in tumor cells, thereby preventing further division and growth, ultimately leading to apoptosis and death. Another example is Purkinje cell-specific promoters (such as PCP-2) and adenovirus vector systems. The expression of PKAc protein or fragments used in Purkinje cells of the cerebellum inhibited the phosphorylation of AKT and ataxin 1, thereby inhibiting the phosphorylation of ataxin 1 and 14-3-3 protein. It can be used to inhibit the binding to the substance. As a result, further progression of the disease is prevented by blocking the important upstream signals necessary for disease progression. AKT knockout mouse Since it does not show the phenotype of cerebellar dysfunction, AKT plays an important role in normal cerebellar function. Since this does not appear to be the case, it is thought that inhibiting AKT in Purkinje cells causes side effects. I can't.

[0144] The invention of the present disclosure will be described in more detail by the following non-limiting examples.

[0145] Example

[0146] In the following examples, the exemplary compounds and / or exemplary compositions refer to some of the compounds in Table 1, as well as all other exemplary compounds described in the present disclosure, and compositions containing them.

[0147] Analysis of the in vitro activity of exemplary compounds: The exemplary compositions were selected from several variants of the ZaTa peptide with significant modifications to improve its activity. The exemplary compounds are cell-permeable peptides that can pass through the cell membrane barrier, inhibit multiple important kinases (including but not limited to AKT, Abl, and P70S6K) in cell proliferation, induce apoptosis, and potently inhibit cell growth. A series of in vitro assays were performed to confirm the activity of the exemplary compounds. More specifically, the following were tested: the ability of each exemplary compound to enter cells, the kinase inhibition profile of each exemplary compound by cell-based kinase assays, its effect on the induction of in vitro apoptosis, and its efficacy against the inhibition of cell growth. More importantly, the efficacy of the exemplary compounds was tested in an animal model of glioblastoma (GBM), using many in vitro and rodent studies to analyze the toxicity, stability, and solubility profiles. The studies described herein confirmed that the activity of the exemplary compounds was well-preserved and highly improved during the lead optimization process. More importantly, these studies demonstrated that the exemplary compounds are significantly superior to existing drugs in terms of efficacy and toxicity profiles.

[0148] ​Inhibition of kinase activity by exemplary compounds: To test the multi-kinase inhibition profiles of exemplary compounds using an in vitro kinase assay, the kinase inhibition profiles of exemplary compounds were tested against approximately 115 different kinases involved in cell proliferation. This kinase profiling includes AKT1, AKT2, p70S6K, and Abl, in addition to these. We demonstrated that various variants of the compound can inhibit several other kinases in the nanomolar range. More specifically, the kinases inhibited within the nanomolar concentration range of different compounds were: AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF), Y340D, Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA (Aurora A), AURKC (O -Lora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK Beta), CDK7 / Cyclin H / MNAT1, and CDC42 BPB (MRCKB). Cell-based kinase assays were designed to check the molecular targets of exemplary compounds in human cancer cells. U251 human glioblastoma cells were treated with several concentrations of exemplary compounds and controls at different time intervals of 20 minutes, 2 hours, and 24 hours to check the phosphorylation of several substrates. The total protein levels were measured at known phosphorylation sites for each kinase. Furthermore, selection Total protein levels of several potential intracellular targets of the identified reads were also measured. Decreased phosphorylation levels of each substrate at known phosphorylation sites would reflect inhibition of kinase activity by the exemplary compounds. Figure 1 shows representative immunoblots from cell-based assays by probing with antibodies recognizing PI3K-P110, or phospho-PDK1 (Ser-241), AKT1, phospho-P53 (Ser-46), phospho-AKT1 (Thr-308), p-CRAF (Ser-259), phospho-Aurora A (Thr-288), or total Aurora A. Different concentrations of vehicle, or Cell-based assays following treatment of U251 human glioblastoma cells with exemplary compounds ranging from 5 μM to 40 μM at different time intervals of 30 minutes, 2 hours, or 24 hours confirmed some of the exemplary compounds' targets by in vitro kinase profiling, demonstrating that the exemplary compounds target several kinases and can inhibit their activity even intracellularly. This experiment also showed that p53 is a downstream target of the exemplary compounds (Figure 1).

[0149] Cellular Penetration Activity of Exemplary Compounds: Several cancer cell lines were tested to study the cell penetration ability of modified exemplary compounds. This experiment showed that the modified peptides could still enter cells. For example, in U251 cells, approximately 10-15% of cells were positively stained with the exemplary compound after 2 hours of treatment with a fluorescently labeled form (image not shown). After 3 days of treatment with the exemplary compound, this percentage increased significantly to 90%. The results of this experiment demonstrate the stability of the cell permeability properties of the exemplary compound during the optimization process.

[0150] Induction of Apoptosis by Exemplary Compounds: The ability of exemplary compounds to induce apoptosis was tested to confirm that the novel modification preserved the peptide's apoptosis-inducing ability. U251 human glioblastoma cells were treated with exemplary compounds at intervals of several hours. Figure 2 shows apoptosis and survival of U251 glioblastoma cells at different time intervals after treatment with exemplary compounds. The same number of U251 human glioblastoma cells (5 x 10⁵) The compounds were plated onto coverslips and treated with either the vehicle (upper panel) or the exemplary compound (lower panel) for 20 minutes, 1, 2, or 3 days. Apoptotic cells were then visualized. To visualize the nuclei of all cells (blue), a fluorescence TUNEL (green) assay is performed. DAPI staining was used for this purpose. As another marker of apoptosis, indirect immunofluorescence staining of cleavage-caspase 3 (red) was performed. Cells treated with the vehicle grew to much higher confluence compared to cells treated with the exemplary compound (compared to the DAPI signal in the upper and lower panels). Confocal images (20X) show the three channels (first three columns) and the merged image (last column) separately at the same Z-step. The image in Figure 2 shows the time-dependent increase in the rate of apoptosis of cells after treatment. After 3 days of treatment, the cells Nearly 100% undergo apoptosis, as shown by both TUNEL (green) and cleavage-caspase 3 (red).

[0151] Quantitative analysis of cell density and apoptosis rate at different time intervals: Equal cell numbers were plated onto coverslips and treated with exemplary compounds and vehicles at different time intervals. Cells were stained with DAPI and TUNEL and analyzed by confocal microscopy. Figure 3(A) shows histogram bars reflecting the average number of total cells (DAPI-positive cells) remaining on the coverslips counted in five separate confocal fields. Figure 3(B) shows the total number of cells remaining on the coverslips (DAPI-positive cells) counted in five separate confocal fields, along with TUNEL-positive cells. This is a histogram bar showing the average percentage of apoptotic cells calculated by dividing the number of sex cells. Cells treated with the vehicle consistently showed higher densities over time and reached near-complete confluence at 72 hours (not shown), while cells treated with the exemplary compound consistently showed a significant decrease in cell density and a time-dependent increase in the number of apoptotic cells over time, with nearly 100% of cells being apoptotic after 72 hours of treatment. It was S (Figure 3).

[0152] Inhibition of cell proliferation by exemplary compounds: The most important activity of exemplary compounds is the inhibition of cell proliferation. Therefore, three exemplary compounds on the cell proliferation rate of several cancer cell types The effects of the compounds were measured. Figure 4 shows the IC50 of exemplary compounds in human breast, lung, blood, and skin cancer cells. Representative plates of several human cancer cell lines are shown in this figure, including metastatic human breast cancer cells (BT-549 and MDA-MB-468), human acute promyelocytic leukemia (HL-60), multiple myeloma cancer cell line (RPMI-8226), and small cell lung cancer cell line ( The cells included DMS-114, a human melanoma cancer cell line (SK-ML-5). Cells were serially diluted from 50 μM to 0.7 μM with three exemplary compounds: vehicle (PBS), or control with other kinase inhibitor compounds in clinical trials (MK-2206), or with compounds in clinical use (imatinib or Gleevec). The material was treated for 3 days. This experiment demonstrates superior efficacy and an IC-50 in the nanomolar range of the exemplary compound compared to an existing class of drugs in the latest stages of market or clinical trials. (Figure 4). An MTT cell proliferation assay was also performed to test the anti-cell proliferation activity of exemplary compounds, and the IC50 was measured in several GBM cell lines using Promega Cell Titer-Glo (registered trademark). The standard assay was performed. Including U87, U251, SNB-75, SF-268, SF-295, and SF-539, The IC50 of exemplary compounds on six different glioblastoma cells ranged from 0.2 to 2.5 μM, and on N2a neuroblastoma cells, it ranged from 0.6 to 1.2 μM. This IC-50 is orders of magnitude superior to Temodar® IC-50 in the same cell line (Sun et al., 2012, Torres et al., 2011, Milano et al., 2009, Kanzawa et al., 2004). ). The IC-50 of Temodar®, the leading brain tumor drug currently on the market, is 421-1021 μM in glioblastoma cells and 602 μM in neuroblastoma cells (Sun et al., 2012, Torres et al., 2011, Milano et al., 2009, Kanzawa et al., 2004). About Gleevec The reported IC-50 levels were 15.7–18.7 μM and 9 μM in glioblastoma and neuroblastoma cell lines, respectively. -13μM (Beppu et al., 2004, Kinsella et al., 2011, Kinsella et al., 2012 ).

[0153] Comparative efficacy studies with minimal use of FDA-approved multi-kinase inhibitors: Exemplary To compare the efficacy of the compound in clinical use with other FDA-approved multi-kinase inhibitors Therefore, the IC50 of exemplary compounds was used in human cell lines of hepatocellular carcinoma (HCC), pancreatic cancer, and metastatic breast cancer. The measurement was performed using sorafenib (approved for HCC), lapatinib (approved for breast cancer), sunitinib, and E. The IC50 was compared with that of rulotinib (approved for pancreatic cancer). The data is for the same FDA-approved indication. Compared to sorafenib, sunitinib, erlotinib, and lapatinib, the exemplary compound demonstrated significantly superior efficacy and a considerably lower IC50.

[0154] Inhibition of Tumor Growth by Exemplary Compounds in Animal Models: To test the efficacy of exemplary compounds in animal models and their ability to locally suppress tumor growth, glioblastoma (GBM), hepatocellular carcinoma (HCC), and metastatic breast cancer flank xenografts were generated in female Nu / Nu mice up to 6 weeks of age. When the tumor volume reached approximately 100 mm3, the animals were randomly divided into two groups of vehicles, approximately 8 animals per group, to receive the compound. Tumor size was measured by caliper before injection and at indicated time intervals after injection, for GBM. The results are shown in Figure 5A, HCC in Figure 5B, and metastatic breast cancer in Figure 5C. During the experiment, animals were closely monitored for weight loss and signs of toxicity. Every three days, animals were administered a single dose of an exemplary compound at 50 mg / kg in a 10 μl volume or 100 μl of saline intratumorally. The drug was administered to the animals. The results of the study are shown in Figure 5. The animals tolerated the 50 mg / kg dose very well, and there were no signs of weight loss or visible toxicity. Tumors in the animals treated with the vehicle continued to grow very rapidly, while tumors in the lead-treated group did not grow and advanced to the control group as early as day 7 of the study. It was clearly much smaller than that. In fact, the tumors almost completely stopped growing, and there was no need to increase the initial dose of the exemplary compound. When the tumors in the control group reached a volume of approximately 3000 mm3, All animals were sacrificed and their tumors were dissected (Figure 5). The control group animals had severe cancer due to advanced cancer. Although the animals appeared to have anorexia and illness, those treated with the exemplary compound appeared healthy, showed no signs of toxicity, and had stable weight. A very significant difference in tumor volume was observed 7 days after the first injection and This difference was observed early on between the control and treatment groups, and it remained highly significant throughout the entire trial (Figures 5A, 5B, and 5C).

[0155] The results of the animal tests were particularly interesting when compared to similar animal efficacy studies of other successful kinase inhibitors used clinically, such as those in Phase III clinical trials of erlotinib (trade name Tarceva), lapatinib (trade names Tykerb and Tyverb), or MK-2206 (an AKT inhibitor). Erlotinib is a receptor tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR), and lapatinib is a dual tyrosine kinase inhibitor that blocks the HER2 / neu and epidermal growth factor receptor (EGFR) pathways. In the study by Hirai et al., the efficacy of MK-2206, erlotinib, and lapatinib was compared individually as single compounds or as combination therapies with each other in a xenograft model. Comparing the results of the tests of the exemplary compounds with the results of the efficacy of MK-2206, erlotinib, and lapatinib in the study by Hirai et al. demonstrated that erlotinib and lapatinib in the study by Hirai et al. showed very excellent efficacy of the exemplary compounds compared to these successful drugs in several respects: a) The exemplary compound showed excellent efficacy as a single composition, and none of the three MK-2206, erlotinib, and lapatinib showed similar efficacy when administered individually. b) The efficacy of the exemplary compound alone was even more excellent than the combination of MK-2206 and erlotinib or the combination of MK-2206 and lapatinib. This is an expected result because the exemplary compound targets a few key kinases simultaneously in cell proliferation. c) The effective dose (50 mg / kg / 3 days) of the exemplary compound in the same animals was much more excellent than the effective doses of MK-2206 (60, 120, and 360 mg / kg / day), erlotinib (50 mg / kg / day), and lapatinib (100 mg / kg / day). The animal tests for the exemplary compound were started at a minimum dose of 50 mg / kg every three days, and this minimum dose was sufficient to completely destroy tumor growth.

[0156] In vitro and cell line stability studies: The stability of the exemplary composition was observed at -20°C, 4°C, and 25°C. And after incubation at different temperatures including 40°C, and in our non-GLP laboratory The samples were studied after several freeze-thaw cycles. After incubation, several samples were drowned and visually examined for physical signs of instability such as aggregation or precipitation. The samples were also tested with GBM cell lines to check for changes in IC50 after incubation. (Similar to the experiment shown in Figure 4). This study showed that the stability of the lead composition in powder form is at -20°C. The peptide was confirmed to survive for several years, at least one year at 4°C, six months at 25°C, and at least 12 weeks at 40°C. It also withstood up to 10 freeze-thaw cycles. The peptide sequence is short and contains amino acids that cause instability (such as Asn or Gln, which tend to deamidate, or Asp or Trp, which tend to hydrolyze and isomerize, or any free Cys, which cause dimerization). Since this hadn't been done before, it wasn't surprising.

[0157] Solubility study: Due to its amphiphilic nature, the exemplary composition is highly water-soluble, at 50 mg / ml They can be readily dissolved in any water-based solution up to a certain concentration. They can also be readily dissolved in many organic solutions commonly used in pharmaceutical formulations.

[0158] In vitro and animal toxicity studies: The toxicity of the exemplary compound, a crucial first step in drug development, was analyzed both in vitro and in rodents, and the results were compared with several other successful kinase inhibitors. These studies demonstrated a superior toxicity profile of the exemplary compound compared to several other kinase inhibitors currently in clinical use.

[0159] As the first in vitro study, the toxicity of an exemplary compound was compared to Gleevec using the 98-well format of the Ames test in bacteria. The Ames test, one of the most frequently applied tests in toxicology, has been used to evaluate bacterial mutagenicity as an endpoint for carcinogenicity. The substance was identified by a Salmonella reverse mutagenesis assay. Almost all new pharmaceuticals and chemicals used in industry are tested using this assay. The results of this test revealed that the exemplary compound was safe at several doses tested in bacteria. The accuracy of this test was validated by testing positive and negative controls, as well as the same doses of Gleevec, as a control assay.

[0160] As another commonly used in vitro toxicity test, we utilized the hERG assay. Numerous structurally and functionally unrelated drugs block hERG potassium channels. hERG channels are involved in the repolarization of cardiac action potentials, and impaired hERG function prolongs ventricular action potentials, extends the QT interval on the electrocardiogram, and increases the risk of potentially fatal ventricular arrhythmias. To reduce the risk of investing resources in drug candidates that fail preclinical safety trials due to QT prolongation, it is crucial to screen compounds for activity on hERG channels early in the lead optimization process. The IC-50 of exemplary compounds was measured against hERG channels using the Predictor® hERG fluorescence polarization assay. This assay allows us to determine which exemplary compounds perform in trials. It was shown that it does not inhibit hERG even at the test concentration.

[0161] The toxicity of exemplary compounds in rodents was also tested by measuring the maximum tolerated dose (MTD) in mice, and its dose was compared to that of successful kinase inhibitors on the market. The single-dose MTD in C57BL / 6 mice ranged from 25 mg / kg to 400 mg / kg, compared to clinical scores. Animals were determined to have body weight as an endpoint. Animals received a single dose of either the exemplary compound or Gleevec, starting at 25 mg / kg on day 1, 50 mg / kg on day 3, 100 mg / kg on day 5, 200 mg / kg on day 7, and 400 mg / kg on day 9. Using this dose escalation study, the exemplary compound was very well tolerated up to a dose of 200 mg / kg, and Gleevec was well tolerated up to a dose of 100 mg / kg. Numerous toxicity tests were conducted in clinics using several other chemotherapeutic agents in C57BL / 6 mice. A study (Aston et al., 2017) confirmed that this dose is an acceptable MTD in C57BL / 6 mice. Comparing this dose to the MTD of Gleevec, and considering that chronic use of the exemplary compound at a single dose of 50 mg / kg / 3 days in Nu / Nu mice is well tolerated and completely disrupts tumor growth, this study confirmed that the drug does not completely disrupt tumor growth. Considering the fact that it was broken down, the exemplary compound would not pass the toxicity test in Phase I clinical trials. It is.

[0162] While this disclosure has been described in relation to what are currently considered to be exemplary actual embodiments, it should be understood that the inventions of this disclosure are not limited to the disclosed embodiments, but rather are intended to cover a variety of modifications and equivalent arrangements that fall within the spirit and scope of the appended claims.

Claims

1. Modified peptides including the following: (i) Amino acid sequence (X)-GRT-(Y)-TLC-(Z), or (ii) An amino acid sequence having at least 40% sequence identity with respect to the amino acid sequence (X)-GRT-(Y)-TLC-(Z), Here X is a natural amino acid, a non-natural amino acid, a chemically modified natural or non-natural amino acid, or acetyl A group, a lipid group, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, a chemically modified natural or non-natural amino acid, or so These are combinations. And; Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an amine group, or a combination thereof.

2. The modified peptide according to claim 1, amino acid sequence: (X 1 )-KGRT-(Y)-TLC-(Z) 、 Here, X 1 This includes natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids, acetyl groups, lipid groups, or combinations thereof.

3. Modified peptide according to claim 1 or 2, amino acid sequence: (X 2 )-VKGRT-(Y)-TLC-(Z)、 Here, X 2 This includes natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids, acetyl groups, lipid groups, or combinations thereof.

4. A modified peptide, amino acid sequence, according to any one of claims 1-3: (X 3 )-RVKGRT-(Y)-TLCGRPE-(Z 1 )、 Here, X 3 Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof, where Z 1 This refers to natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids, amine groups, or combinations thereof.

5. A modified peptide, amino acid sequence, according to any one of claims 1-4: (X 4 )-KRVKGRT-(Y)-TLCGRPE-(Z 1 )、 Here, X 4 Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof, where Z 1 This refers to natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids, amine groups, or combinations thereof.

6. The modified peptide according to any one of claims 1-5 has the amino acid sequence: (X 5 )-RVKGRT-(Y)-TLCGRPE-(Z 1 )、 Here, X 5 Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof, where Z 1 This refers to natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids, amine groups, or combinations thereof.

7. A modified peptide, amino acid sequence, according to any one of claims 1-6: (X 7 )-V-(X 8 )-GRT-(Y)-TLC-(Z)、 Here, X 7 X is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof, where X 8 These are natural amino acids, unnatural amino acids, chemically modified natural or unnatural amino acids, or combinations thereof. ru.

8. A modified peptide, amino acid sequence, according to any one of claims 1-7: (X 9 )-KV-(X 8 )-GRT-(Y)-TLC-(Z)、 Here, X 9 X is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an acetyl group, a lipid group, or a combination thereof, where X 8 These are natural amino acids, unnatural amino acids, chemically modified natural or unnatural amino acids, or combinations thereof.

9. A modified peptide according to any one of claims 1-9: X contains an acetyl group, a lauroyl group, or a palmitoyl group located at its terminal end; Y is 1-Nal; Z contains an amino group located at its terminal end.

10. Modified peptides having the formula: (X)-(seq1)-(Y)-(seq2)-(Z) or an amino acid sequence (X)-(seq1)-(Y)-(seq2)-(Z) having at least 40% sequence identity with respect to the amino acid sequence, Here: seq1 is GRT, KGRT, VKGRT, RVKGRT, KRVKGRT, (Orn)-RVKGRT, or AKRVKGRT; seq2 is TLC, TLCG, TLCGR, TLCGRPE, TLCGRPEY, or TLCGRPE-(4-Cl-Phe); X is a natural amino acid, a non-natural amino acid, a chemically modified natural or non-natural amino acid, or acetyl A group, a lipid group, or a combination thereof; Y is a natural amino acid, a non-natural amino acid, a chemically modified natural or non-natural amino acid, or so These are combinations, Z is a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid, an amine group, or a combination thereof.

11. A modified peptide according to any one of claims 1 to 10, wherein X comprises an acetyl group or lipid group located at its terminal end.

12. A modified peptide according to any one of claims 1 to 11, wherein the lipid group is a C6 to C20 lipid group. That is the case.

13. A modified peptide according to any one of claims 1 to 12, wherein the lipid group is a lauroyl group or a palmitoyl group.

14. A modified peptide according to any one of claims 1-13, wherein Y is 1-Nal.

15. A modified peptide according to any one of claims 1 to 14, wherein Z comprises an amino group located at its terminal end.

16. A modified peptide according to any one of claims 1-15, wherein the non-natural amino acid is ornithine, naphthylalanine, 4-chlorophenylalanine, or a combination thereof.

17. A dimer of the modified peptide according to any one of claims 1 to 16.

18. The dimer according to claim 17, wherein the dimer contains a disulfide bond.

19. The dimer according to claim 17 or 18, wherein the dimer is a homodimer or a heterodimer.

20. Modified peptide having the following formula: (i) The following structure: Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 (Cys-Cys dimer)、 Lauroyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 、 Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 (Cys-Cys dimer)、 Palmitoyl-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 、 Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 (Cys-Cys dimer)、 Ac-(Orn)-RVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 、 Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 、 Ac-AKRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 (Cys-Cys dimer)、 Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 、 Ac-KRVKGRT-(1-Nal)-TLCGRPE-(4-Cl-Phe)-NH 2 (Cys-Cys dimer)、 Ac-KGRT-(1-Nal)-TLC-NH 2 、 Ac-KGRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer)、 Ac-VKGRT-(1-Nal)-TLC-NH 2 、 Ac-VKGRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer)、 Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH 2 、 Ac-V-(Orn)-GRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer)、 Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH 2 、 Ac-V-(Orn)-GRT-(1-Nal)-TLCG-NH 2 (Cys-Cys dimer)、 Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH 2 、 Ac-V-(Orn)-GRT-(1-Nal)-TLCGR-NH 2 (Cys-Cys dimer)、 Ac-(Orn)-GRT-(1-Nal)-TLC-(4-Cl-Phe)-NH 2 、 Ac-(Orn)-GRT-(1-Nal)-TLC--(4-Cl-Phe)-NH 2 (Cys-Cys dimer)、 Ac-(Orn)-GRT-(1-Nal)-TLC-NH 2 、 Ac-(Orn)-GRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer)、 Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH 2 、 Ac-KV-(Orn)-GRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer)、 Ac-KVKGRT-(1-Nal)-TLC-NH 2 、 Ac-KVKGRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer)、 Ac-RVKGRT-(1-Nal)-TLC-NH 2 ,or Ac-RVKGRT-(1-Nal)-TLC-NH 2 (Cys-Cys dimer); or (ii) A composition having 40% peptide sequence identity with respect to the composition described in claim 20.

21. At least one modified peptide according to any one of claims 1 to 20, and pharmaceutically acceptable A pharmaceutical composition comprising a carrier or excipient.

22. An isolated nucleic acid encoding a modified peptide according to any one of claims 1 to 20.

23. The isolated nucleic acid according to claim 22, further comprising at least one additional polynucleotide component adjacent to the nucleic acid encoding the modified peptide, wherein the additional polynucleotide is located at the 5' end or the 3' end, or both, of the nucleic acid encoding the modified peptide.

24. A vector comprising the nucleic acid according to claim 22, operably linked to one or more regulatory nucleic acid sequences.

25. A host cell comprising the vector according to claim 22.

26. The host cell according to claim 25, wherein the host cell is a prokaryotic or eukaryotic cell.

27. A device for local delivery of a therapeutic agent, comprising a carrier and the pharmaceutical composition according to claim 21, wherein the carrier enables controlled release of a modified peptide.

28. At least one selected from PKAc, a PKAc fragment, and the one described in claim 1-20 A carrier for targeted delivery of therapeutic or contrast agents to cells or tissues, comprising a variant PKAc fragment having the amino acid sequence of a modified peptide.

29. From the group consisting of protein kinase B (AKT1), p70S6K, and Abl, AKT1 (PKB alpha), AKT2 (PKB beta), MAP3K8 (COT), MST4, AURKB (Aurora B), ROCK1, RPS6KB1 (p70S6K), CDC42 BPA (MRCKA), BRAF, RAF1 (cRAF) Y340D Y341D, SGK (SGK1), MAP4K4 (HGK), AURKA (Aurora A), AURKC (Aurora C), BRAF V599E, CHEK1 (CHK1), GSG2 (Haspin), CHEK2 (CHK2), FGR, IKBKB (IKK beta), CDK7 / cyclin H / MNAT1, and CDC42 BPB (MRCKB) A method for inhibiting the activity of at least one selected enzyme: the method comprises contacting at least one selected from the enzymes with any of (i) an enzyme having PKAc, a PKAc fragment, and a variant PKAc fragment having the amino acid sequence of the modified peptide described in claim 1, or (ii) the carrier described in claim 28, or (iii) the pharmaceutical composition described in claim 21, thereby inhibiting the activity of the enzyme.

30. A method for inhibiting cell proliferation, comprising contacting cells with the pharmaceutical composition according to claim 21, which inhibits cell proliferation.

31. A method for preventing or treating cancer, comprising administering an effective amount of the pharmaceutical composition according to claim 21 to a patient who has cancer or is at risk of having cancer, wherein the cancer is prevented or treated.

32. A method for preventing or treating a neurodegenerative disease or disorder, comprising administering an effective amount of the pharmaceutical composition described in claim 21 to a patient who has or is at risk of having a neurodegenerative disease or disorder.

33. Methods for preventing or treating immunodeficiency disorders, including but not limited to the following: AIDS, leukemia, lymphoma, viral diseases, such as hepatitis, multiple myeloma, ataxia telangiectasia, Chediak-Higashi syndrome, combined immunodeficiency, complement deficiency, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion disorder, panhypogammaglobulinemia, Bruton's disease, congenital agammaglobulinemia, selective IgA deficiency, and Furthermore, in the case of Wiscott-Aldrich syndrome, this method comprises administering an effective amount of the pharmaceutical composition described in claim 21 to a patient in need.

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