Ad36 e4orf1 peptide fragments as anti-diabetic agents

Abstract

Embodiments of the present disclosure relate to a composition comprising at least one early gene 4 open reading frame 1 (E4orf1) peptide fragment. The E4orf1 peptide fragment includes, but is not limited to, SEQ ID NO: 1 or a sequence having at least 65% sequence identity to SEQ ID NO: 1; SEQ ID NO: 2 or a sequence having at least 65% sequence identity to SEQ ID NO: 2; or a combination thereof. Other embodiments of the present disclosure relate to methods of modulating glucose uptake by a cell by combining the cell with the composition described herein. Further embodiments of the present disclosure relate to methods of treating or preventing a metabolic disorder in a subject by administering the composition described herein to the subject.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 543,338, filed October 10, 2023. The entire contents of the above application are incorporated herein by reference.

[0003] Serial Public Declaration

[0004] Pursuant to 37 CFR § 1.834, the applicant has submitted a sequence list in XML format (“SequenceListing”). The text file containing the sequence list is named “AF13368.P059WO.xml”. The sequence list was created on October 10, 2024. The sequence list is 6,000 bytes in size. The applicant hereby references the materials included in the sequence list. Background Technology

[0005] There is an urgent need for more effective drugs to treat or prevent metabolic disorders. Many embodiments of this disclosure are designed to address these needs. Summary of the Invention

[0006] In some embodiments, embodiments of this disclosure relate to a composition comprising at least one early gene 4 open reading frame 1 (E4orf1) peptide fragment. In some embodiments, the E4orf1 peptide fragment includes, but is not limited to, REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence having at least 65% sequence identity with SEQ ID NO: 1; YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 2) or a sequence having at least 65% sequence identity with SEQ ID NO: 2; or combinations thereof.

[0007] Other embodiments of this disclosure relate to methods for regulating cellular glucose uptake by associating cells with the compositions described herein. Further embodiments of this disclosure relate to methods for treating or preventing metabolic disorders in a subject by administering the compositions described herein to the subject.

[0008] The methods disclosed herein can be used to treat or prevent various metabolic disorders in subjects. For example, in some embodiments, the metabolic disorders include, but are not limited to, prediabetes, type 1 diabetes (T1D), type 2 diabetes (T2D), insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or a combination thereof. Attached Figure Description

[0009] Figure 1A This study elucidates how the early gene 4 open reading frame 1 (E4orf1) bypasses the proximal insulin signaling pathway but activates the distal insulin signaling pathway.

[0010] Figure 1B The amino acid sequence of E4orf1 is shown, in which the PDZ domain-binding (PBM) motif is underlined.

[0011] Figure 2A-2C Empty nanoparticles are shown. Figure 2A Nanoparticles treated with a 25-amino acid E4orf1 peptide fragment ( Figure 2B ) and nanoparticles treated with E4orf1 ( Figure 2C Nanoparticles were imaged using transmission electron microscopy (TEM).

[0012] Figure 3 Western blots show the expression of phosphorylated (p)-Akt (approximately 60 kDa) and total Akt (approximately 60 kDa) in 3T3L1 cells treated with empty nanoliposomes (empty NP), E4orf1 nanoliposomes (E4 NP), and 100 nM insulin (insulin) for 24 hours. The bar chart shows the p-Akt level normalized to total Akt, which was significantly higher in the E4 NP group than in the empty NP group (ANOVA, P). 0.05).

[0013] Figures 4A-4B Western blots show the expression of phosphorylated (p)-Akt (approximately 60 kDa) and total Akt (approximately 60 kDa) in 3T3-L1 cells treated with 70 μL of empty NP, E4orf1 NP, P5 NP, P5L NP, P25 NP, and 100 nM insulin (insulin) for 12 and 24 hours. The bar charts show p-Akt levels normalized to total Akt levels, which were not significantly different in the peptide NP treatment groups (P5, P5L, P25) compared to the empty NP treatment group. However, Akt activation was significantly higher in the E4 NP group compared to both the empty NP group and the peptide NP treatment group (ANOVA, P < 0.05). 0.05).

[0014] Figures 5A-5B Western blots show the expression of phosphorylated (p)-Akt (approximately 60 kDa) and total Akt (approximately 60 kDa) in 3T3L1 cells treated with 70 μL of empty NP, E4orf1 NP, P25 NP, and 100 nM insulin (insulin) for 48 and 72 hours. The bar charts show p-Akt levels normalized to total Akt levels, which were not significantly different in the P25 NP and E4orf1 NP groups compared to the empty NP group (ANOVA, P < 0.05). 0.05).

[0015] Figures 6A-6B Western blots showing the expression of phosphorylated (p)-Akt (approximately 60 kDa) and total Akt (approximately 60 kDa) in 3T3-L1 cells treated with 70 μL of empty NP, P5 NP, and P25 NP for 12 hours. Figure 6A The bar chart shows the p-Akt levels standardized to the total Akt level, which are significant in the p25 NP treatment group compared to the empty NP treatment group (ANOVA, p). 0.05). Figure 6B Subsequent experiments showed that the P25 NP treatment group had no significant difference from the empty NP treatment group.

[0016] Figures 7A-7B The results of P25 NP-mediated glucose uptake are shown. Figure 7A The original data before standardization are shown, demonstrating a significant difference between the P25 treatment group and the empty NP treatment group after 12 hours (ANOVA, P). 0.05). Figure 7B Additional data are provided, showing that at 12 hours after standardization, there was no significant difference between the P25 NP treatment group and the empty NP treatment group (ANOVA, P). 0.05).

[0017] Figures 8A-8B Western blots show the expression of phosphorylated (p)-Akt (approximately 60 kDa) and total Akt (approximately 60 kDa) in 3T3-L1 cells treated with 0.208 mM P70 Ad36E4orf1 peptide for 12 and 24 hours. The bar charts show p-Akt levels normalized to total Akt levels, which were significant in cells treated with peptide P70-NP for 12 hours compared to cells treated with empty NP. Detailed Implementation

[0018] It should be understood that the foregoing general description and the following detailed description are illustrative and explanatory, and do not limit the subject matter for which patent protection is claimed. In this application, the use of the singular includes the plural, the words “a” or “an” mean “at least one / an,” and the use of the word “or” means “and / or,” unless otherwise specified. Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not restrictive. Meanwhile, unless otherwise specified, terms such as “element” or “component” include elements or components comprising one unit as well as elements or components comprising more than one unit.

[0019] The section headings used herein are for organizational purposes and should not be construed as limiting the content. All documents or portions thereof, including but not limited to patents, patent applications, articles, books, and treaties, cited in this application are incorporated herein by reference in their entirety for any purpose. In the event of any conflict between the definition of a term in one or more combined documents and similar materials and the definition of a term in this application, the definition in this application shall prevail.

[0020] Metabolic disorders are a significant public health problem. For example, diabetes is one of the most common complex metabolic disorders, with heterogeneous pathogenesis. Type 1 diabetes (T1D) is characterized by a defect in insulin secretion from pancreatic β-cells, while type 2 diabetes (T2D) reflects impaired insulin response in insulin-sensitive tissues.

[0021] It is estimated that as of 2019, approximately 9.3% (463 million people) worldwide were diagnosed with diabetes, and this figure is projected to rise to 10.2% (578 million people) by 2030 and reach 12.4% (700 million people) by 2045. According to the 2022 U.S. National Diabetes Statistics Report, more than 130 million people of all ages in the United States have diabetes or prediabetes. Of these, more than 90% have type 2 diabetes. Therefore, the prevalence of diabetes continues to rise, becoming a serious global health challenge.

[0022] Insulin resistance is one of the common characteristics of type 2 diabetes mellitus (T2D). For example... Figure 1A As shown, the insulin signaling pathway mainly consists of metabolic and mitotic pathways. Most of the metabolic effects of insulin are related to the PI3K / AKT pathway. Therefore, the pathogenesis of insulin resistance is primarily related to the PI3K / AKT pathway. Typically, the PI3K / AKT pathway is divided into proximal and distal insulin signaling pathways. Insulin binds to the insulin receptor on the cell membrane, followed by autophosphorylation of insulin receptor substrate 1 (IRS1) and insulin receptor substrate 2 (IRS2), thereby activating the PI3K / AKT pathway.

[0023] Phosphorylation of Akt in the PI3K / AKT pathway activates Akt, leading to the translocation of glucose transporter 4 (Glut 4) to the cell membrane for glucose uptake. Conformational changes in IRS1 and IRS2 suggest a proximal signaling pathway, while PI3K / AKT and Glut 4 suggest a distal signaling pathway. However, in insulin resistance, the proximal signaling pathway fails to transmit the signal that prompts cells to take up glucose from the bloodstream. Furthermore, most current antidiabetic drugs, such as insulin secretagogues, insulin analogs, or insulin sensitizers, focus on activating the proximal insulin signaling pathway to enhance cellular glucose uptake.

[0024] Therefore, there is an urgent need for more effective drugs to treat or prevent metabolic disorders. In particular, there is a need for more effective drugs to activate distal insulin signaling pathways in the presence or absence of insulin. Many embodiments of this disclosure are designed to address these needs.

[0025] Composition

[0026] In some embodiments, embodiments of this disclosure relate to a composition comprising at least one early gene 4 open reading frame 1 (E4orf1) peptide fragment. In some embodiments, the E4orf1 peptide fragment includes, but is not limited to, REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence having at least 65% sequence identity with SEQ ID NO: 1; YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 2) or a sequence having at least 65% sequence identity with SEQ ID NO: 2; or combinations thereof. As described in more detail herein, the compositions of this disclosure can have various embodiments.

[0027] E4orf1 peptide fragment

[0028] The compositions disclosed herein may comprise various E4orf1 peptide fragments. For example, in some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 65% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 70% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 75% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 85% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 95% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the E4orf1 peptide fragment comprises SEQ ID NO: 1.

[0029] In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 65% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 70% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 75% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 80% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 85% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 90% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 95% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises a sequence having at least 99% sequence identity with SEQ ID NO: 2. In some embodiments, the E4orf1 peptide fragment comprises SEQ ID NO: 2.

[0030] delivery agent

[0031] In some embodiments, the E4orf1 peptide fragment of this disclosure may bind to a delivery agent. The E4orf1 peptide fragment of this disclosure may bind to a variety of delivery agents. For example, in some embodiments, the delivery agent includes, but is not limited to, particles, nanoparticles, lipid-based nanoparticles, liposomes, nanoliposomes, or combinations thereof.

[0032] The E4orf1 peptide fragment disclosed herein can bind to a delivery agent in a variety of ways. For example, in some embodiments, the E4orf1 peptide fragment is encapsulated within the delivery agent. In some embodiments, the E4orf1 peptide fragment is located on the surface of the delivery agent. In some embodiments, the E4orf1 peptide fragment is non-covalently linked to the delivery agent.

[0033] Composition components and forms

[0034] The compositions disclosed herein may contain a variety of other components. For example, in some embodiments, the compositions disclosed herein include at least one solubilizer. In some embodiments, the solubilizer includes, but is not limited to, polyethylene glycol, glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylene glyceryl ester, polyoxyethylene glyceryl ester, polysorbate, dehydrated sorbitan monooleate, hydroxypropyl-β-cyclodextrin (HPCD), polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene hydroxystearate, or combinations thereof.

[0035] The compositions disclosed herein can be in various forms. For example, in some embodiments, the compositions disclosed herein may be in liquid form. In some embodiments, the compositions disclosed herein may be in solid form. In some embodiments, the compositions disclosed herein may be in lyophilized form.

[0036] use

[0037] The compositions disclosed herein can have a variety of uses. For example, in some embodiments, the compositions disclosed herein are suitable for treating or preventing metabolic disorders in subjects. In some embodiments, said metabolic disorders include, but are not limited to, prediabetes, type 1 diabetes (T1D), type 2 diabetes (T2D), insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or a combination thereof.

[0038] Methods to regulate cellular glucose uptake

[0039] Other embodiments of this disclosure relate to methods for regulating cellular glucose uptake by binding cells to the compositions described herein. The compositions of this disclosure can bind to a variety of cells. For example, in some embodiments, the cells include adipocytes, muscle cells, or hepatocytes.

[0040] The compositions disclosed herein can produce a variety of effects on cells. For example, in some embodiments, the compositions disclosed herein can increase cellular glucose uptake. In some embodiments, the compositions disclosed herein can increase cellular glucose uptake independently of the proximal insulin signaling pathway. In some embodiments, the compositions disclosed herein can lead to increased glucose uptake by adipocytes, muscle cells, or hepatocytes.

[0041] The compositions disclosed herein can bind to cells in various ways. For example, in some embodiments, the binding of cells to the compositions of this disclosure occurs in vitro. In some embodiments, the binding of cells to the compositions of this disclosure occurs in vivo.

[0042] Methods for treating or preventing metabolic disorders

[0043] Other embodiments of this disclosure relate to methods for treating or preventing metabolic disorders in a subject by administering the compositions described herein to the subject. As described in more detail herein, the methods of this disclosure can have various embodiments.

[0044] Metabolic disorders

[0045] The methods disclosed herein can be used to treat or prevent various metabolic disorders in subjects. For example, in some embodiments, the metabolic disorders include, but are not limited to, prediabetes, type 1 diabetes (T1D), type 2 diabetes (T2D), insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or combinations thereof. In some embodiments, the metabolic disorder includes type 1 diabetes (T1D). In some embodiments, the metabolic disorder includes type 2 diabetes (T2D).

[0046] Beyond theoretical limitations, the compositions of this disclosure can treat or prevent metabolic disorders in a variety of ways. For example, in some embodiments, the compositions of this disclosure can lower endogenous insulin levels in the blood. In some embodiments, the compositions of this disclosure can improve blood glucose levels by lowering high glucose levels in the blood, reducing the expected postprandial rise in blood glucose or the duration of the rise in blood glucose, or reducing hepatic glucose output. In some embodiments, the compositions of this disclosure can prevent increases in blood insulin levels.

[0047] In some embodiments, the compositions of this disclosure can increase glucose uptake by target cells. In some embodiments, the compositions of this disclosure can increase cellular glucose uptake, and this increase is independent of the proximal insulin signaling pathway. In some embodiments, the compositions of this disclosure can lead to increased glucose uptake by adipocytes, muscle cells, hepatocytes, or combinations thereof.

[0048] Dosage / Administration

[0049] The compositions disclosed herein can be administered to the subject in a variety of ways. For example, in some embodiments, the administration is performed via methods including, but not limited to, electroporation, transfection, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, injection, intrathecal injection, intra-articular administration, local administration, central administration, peripheral administration, aerosol-based administration, nasal administration, intranasal administration, transmucosal administration, percutaneous administration, parenteral administration, or combinations thereof. In some embodiments, the administration is performed via methods including, but not limited to, oral administration, intramuscular administration, intranasal administration, subcutaneous administration, percutaneous administration, intravenous administration, or combinations thereof.

[0050] object

[0051] The compositions disclosed herein can be given to a variety of subjects. For example, in some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal includes, but is not limited to, horses, rabbits, mice, rats, pigs, sheep, cattle, dogs, or cats. In some embodiments, the non-human mammal is a domesticated animal, such as a dog or cat. In some embodiments, the subject suffers from a metabolic disorder. In some embodiments, the subject is susceptible to metabolic disorders.

[0052] Other implementation methods

[0053] More specific embodiments of this disclosure and experimental data supporting these embodiments will now be referenced. However, the applicant states that the following disclosure is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0054] Example 1. Ad36E4orf1 peptide as an antidiabetic therapeutic drug

[0055] In previous research, the applicant identified a 125-amino acid protein encoded by the E4orf1 gene (i.e., early gene 4 open reading frame 1) from human adenovirus (Ad36). This protein has shown anti-hyperglycemic effects in vitro (preadipocytes, adipocytes, myoblasts, and / or skeletal muscle) and in animal models. This protein (Ad36E4orf1) has the potential to lower blood glucose levels and increase cellular glucose uptake, even in the presence of insulin resistance, by bypassing impaired proximal insulin signaling pathways and preferentially activating distal insulin signaling pathways.

[0056] Therefore, the insulin-independent action of Ad36E4orf1 makes it a novel potential drug for the treatment of type 1 and type 2 diabetes. Furthermore, this protein exhibits inhibitory effects on hepatic glucose output and lipid accumulation both in vivo and in vitro, suggesting that Ad36E4orf1 has promising potential in preventing the progression of hepatic steatosis. Thus, this protein provides a research opportunity for the development of novel antidiabetic drugs with multiple potential advantages.

[0057] The Ad36E4orf1 protein is not naturally expressed in humans. Therefore, this protein is preferably bound to a delivery system to enter cells and exert its function. The applicant's laboratory previously constructed a lipid-based nanoparticle that successfully delivered the E4orf1 protein in a mouse-derived preadipocyte (3T3-L1) cell line, exerting an anti-diabetic effect via distal cell signaling pathways. However, the purified E4orf1 protein contains a glutathione transferase (GST) tag, which is commonly used in protein purification. Removing this tag is challenging because it affects total protein yield. Furthermore, the use of tagged proteins can trigger immune responses in vivo (e.g., opsonization of serum proteins and clearance by the mononuclear phagocytic system), which is undesirable.

[0058] Therefore, the applicant aims to establish a peptide of shorter amino acid length based on the Ad36E4orf1 protein sequence as an alternative method in this embodiment. The Ad36E4orf1 protein sequence consists of a PDZ domain-binding (PBM) motif, which is essential for its function. Figure 1B In fact, the applicant's previous experiments showed that Ad36E4orf1 requires a PBM region to function. Therefore, the applicant designed peptides of various amino acid lengths containing a PBM sequence.

[0059] Example 1.1. Synthesis of E4orf1 peptide fragment containing PBM domain

[0060] The applicant's primary objective is to determine whether nanoparticle-mediated in vitro and in vivo delivery of E4orf1 peptide fragments can mimic the function of the complete Ad36E4orf1 protein. Compared to recombinant proteins, peptide-based drugs offer several advantages, such as higher site selectivity, higher specificity, lower immunogenicity, and greater therapeutic efficacy. The applicant tested peptides of various amino acid lengths (5, 10, and 25 amino acid sequences) from the E4orf1 protein.

[0061] Table 1 summarizes the different E4orf1 peptide fragments tested in this embodiment.

[0062]

[0063] Table 1. Tested E4orf1 peptide fragments (PBM domains are underlined).

[0064] As shown in Table 1, the P5 peptide fragment has a five-amino acid sequence. The molecular weight of this peptide is 515.35 Daltons. The P5L peptide has the same sequence as the P5 peptide, plus four additional lysine residues. This synthetic peptide is inherently hydrophilic. The molecular weight of this peptide is 1028.3 Daltons. Furthermore, the molecular weight of the P25 peptide is 2773.33 Daltons. These shorter peptides, which do not carry a GST tag, would be shortened versions of the full-length E4orf1 protein, and the applicant anticipates they will be less immunogenic and more cost-effective.

[0065] Example 1.2. Characterization of liposomes

[0066] Nanoparticles are clinically relevant for the delivery of Ad36E4orf1. Liposomes are well-known nanoparticles, and liposomal drug products were approved by the U.S. Food and Drug Administration (FDA) in 1995 for the treatment of Kaposi's sarcoma associated with chemotherapy-resistant acquired immunodeficiency syndrome (AIDS). Currently, liposomes are used in immunology, dermatology, vaccine adjuvants, ophthalmic diseases, brain targeting, infectious diseases, and oncology. Liposome-mediated drug delivery systems also inhibit rapid clearance of liposomes by controlling size, charge, and surface hydration. Furthermore, liposomes protect the encapsulated material from physiological degradation. In addition, liposomes offer higher therapeutic efficacy, optimal biocompatibility, and optimal safety. Therefore, in the delivery of Ad36E4orf1 and its peptide fragments, nanoparticle technology can protect peptides and proteins from degradation, increase their half-life, control protein release, and target different tissues in vivo.

[0067] To analyze and confirm the morphology and size of the nanoparticles, the applicant performed transmission electron microscopy (TEM) imaging on the nanoparticles. Figure 2A-2C Empty nanoparticles are shown. Figure 2ANanoparticles treated with peptide-25 Figure 2B ) and nanoparticles treated with E4orf1 protein ( Figure 2C ).like Figure 2A-2C As shown, the spherical shape of the nanoparticles was preserved when encapsulated with 25aa peptide and 125aa Ad36E4orf1 protein, consistent with the applicant's previous observations. The nanoparticle sizes extracted from the images are in the 70-100 nm range. TEM imaging confirmed the applicant's desired morphology and size distribution of the nanoparticles. Figure 2A-2C ).

[0068] Example 1.3. Activation of insulin signal transduction via Ad36E4orf1

[0069] The applicant investigated whether the 125aa Ad36E4orf1 protein could be delivered via nanoliposome encapsulation to examine Akt phosphorylation as a marker of activation of the distal insulin signaling pathway. Figure 3 As shown, compared to control cells treated with empty nanoliposomes (empty group), 3T3-L1 cells treated with Ad36E4orf1 nanoliposomes (E4orf1-NP) showed a significant increase in pAkt levels after 24 hours of treatment. Positive control cells treated with insulin also showed significant phosphorylation of Akt. Figure 3 ).

[0070] The applicant then examined the ability of 50 uM Ad36E4orf1 peptides P5L and P25 to enhance Akt phosphorylation in 3T3-L1 cells. For example... Figures 4A-4B As shown, treatment with P5L or P25 encapsulated in nanoliposomes for 12 or 24 hours did not increase Akt phosphorylation compared to empty control cells. Furthermore, treatment with 50 μM peptide 25 (P25) encapsulated in nanoliposomes for 48 or 72 hours did not increase Akt phosphorylation compared to empty control cells. Figures 5A-5B ).

[0071] The applicant then determined the dose-dependent effects of P5 and P25 (0.25 ug / uL and 0.50 ug / uL) on Akt phosphorylation in 3T3-L1 cells. Figures 6A-6B As shown, compared to empty-treated control cells, nanoliposome-encapsulated 0.25 μg / μL P25 cells exhibited significant Akt phosphorylation after 12 hours of treatment. Figure 6A However, subsequent experiments did not show the results previously observed. Figure 6B ).

[0072] Example 1.4. Glucose Uptake Test

[0073] Next, the applicant conducted a glucose uptake assay to determine the ability of Ad36E4orf1 P25 to enhance cellular glucose uptake. Mouse 3T3-L1 cells were treated with nanoliposomes encapsulated with 0.25 μg / μL P25 for 12 hours, followed by exposure to 3H radiolabeled glucose. Glucose content in cell lysates was measured using a scintillation counter. Figures 7A-7B As shown, compared to control cells with no treatment, cells treated with Ad36E4orf1 P25 exhibited increased cellular glucose levels. Figure 7A However, when these values ​​are standardized to the amount of protein, there is no difference. Figure 7B In summary, the data indicate that the Ad36E4orf1 25aa peptide (P25) can enhance cellular glucose uptake by activating insulin-independent distal insulin signaling.

[0074] Example 2. Activation of insulin signal transduction by the 70-amino acid Ad36E4orf1 peptide.

[0075] In Example 1, the applicant reported that the Ad36E4orf1 25aa peptide (P25) can enhance cellular glucose uptake by activating insulin-independent distal insulin signaling. To further investigate other Ad36E4orf1 peptides, the applicant tested the effects of peptides with a length of 70 amino acids, and the results are shown in Table 2.

[0076]

[0077] Table 2. E4orf1 peptide fragments tested in Example 2 (PBM domains are underlined).

[0078] As shown in Table 2, P70 is a synthetic peptide with a 70-amino acid sequence. The molecular weight of this peptide is 8083.38 Daltons.

[0079] Mouse 3T3-L1 preadipocytes were treated for 12 or 24 hours with either 0.208 μM P70 Ad36E4orf1 peptide-encapsulated nanoliposomes (peptide group) or empty nanoliposomes (empty group). Figure 8A As shown, compared to control cells treated with empty control, 3T3-L1 cells treated with P70Ad36E4orf1 peptide for 12 hours showed a significant increase in pAkt protein expression. Cells treated with P70Ad36E4orf1 peptide for 24 hours did not show an increase in pAkt expression. Figure 8A ).

[0080] Next, the applicant examined the expression of the Ad36E4orf1 protein in these cells. Protein lysates from transgenic mice expressing 125aaAd36E4orf1 protein were used as a positive control. After treating cells with the P70 Ad36E4orf1 peptide for 12 hours, the cells showed protein expression, while the empty control cells showed no protein expression, confirming the protein translation of this peptide. Figure 8B ).

[0081] Without further elaboration, it is believed that those skilled in the art will fully utilize the content of this invention through the description herein. The embodiments described herein should be understood as illustrative and not in any way limiting the remainder of this disclosure. While embodiments have been shown and described, those skilled in the art can make many variations and modifications thereto without departing from the spirit and teachings of the invention. Therefore, the scope of protection is not limited by the foregoing description, but only by the claims, including all equivalents of the subject matter of the claims. All patent, patent application, and publication disclosures listed herein are incorporated herein by reference, and within this scope, their provision or other details are consistent with and supplement to those described herein.

Claims

1. A composition comprising at least one early gene 4 open reading frame 1 (E4orf1) peptide fragment, wherein the E4orf1 peptide fragment is selected from the group consisting of: REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence having at least 65% sequence identity with SEQ ID NO: 1; YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 2) or a sequence having at least 65% sequence identity with SEQ ID NO: 2; Or a combination thereof.

2. The composition of claim 1, wherein the E4orf1 peptide fragment is bound to a delivery agent.

3. The composition of claim 2, wherein the delivery agent is selected from the group consisting of particles, nanoparticles, lipid-based nanoparticles, liposomes, nanoliposomes, or combinations thereof.

4. The composition of claim 2, wherein the E4orf1 peptide fragment is encapsulated in the delivery agent.

5. The composition of claim 1, wherein the E4orf1 peptide fragment comprises SEQ ID NO: 1 or a sequence having at least 65% sequence identity with SEQ ID NO:

1.

6. The composition of claim 1, wherein the E4orf1 peptide fragment comprises SEQ ID NO:

1.

7. The composition of claim 1, wherein the E4orf1 peptide fragment comprises SEQ ID NO: 2 or a sequence having at least 65% sequence identity with SEQ ID NO:

2.

8. The composition of claim 1, wherein the E4orf1 peptide fragment comprises SEQ ID NO:

2.

9. The composition of claim 1, wherein the composition is suitable for treating or preventing metabolic disorders in a subject.

10. The composition of claim 9, wherein the metabolic disorder is selected from the group consisting of: prediabetes, type 1 diabetes (T1D), type 2 diabetes (T2D), insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or a combination thereof.

11. A method for treating or preventing metabolic disorders in a subject, the method comprising: The composition is given to a subject, wherein the composition comprises at least one early gene 4 open reading frame 1 (E4orf1) peptide fragment, wherein the E4orf1 peptide fragment is selected from the group consisting of: REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence having at least 65% sequence identity with SEQ ID NO: 1; YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 2) or a sequence having at least 65% sequence identity with SEQ ID NO: 2; or Its combination.

12. The method of claim 11, wherein the administration is performed via a method selected from the group consisting of: electroporation, transfection, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, injection, intrathecal injection, intra-articular administration, local administration, central administration, peripheral administration, aerosol-based administration, nasal administration, intranasal administration, transmucosal administration, transdermal administration, parenteral administration, or combinations thereof.

13. The method of claim 11, wherein the administration is performed via a method selected from the group consisting of oral administration, intramuscular administration, intranasal administration, subcutaneous administration, percutaneous administration, intravenous administration, or a combination thereof.

14. The method of claim 11, wherein the metabolic disorder is selected from the group consisting of: prediabetes, type 1 diabetes (T1D), type 2 diabetes (T2D), insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or a combination thereof.

15. The method of claim 11, wherein the composition reduces the level of endogenous insulin in the blood.

16. The method of claim 11, wherein the composition prevents an increase in blood insulin levels.

17. The method of claim 11, wherein the object is a person.

18. The method of claim 11, wherein the E4orf1 peptide fragment is bound to the delivery agent.

19. The method of claim 18, wherein the delivery agent is selected from the group consisting of particles, nanoparticles, lipid-based nanoparticles, liposomes, nanoliposomes, or combinations thereof.

20. The method of claim 11, wherein the E4orf1 fragment is encapsulated in the delivery agent.

21. The method of claim 11, wherein the E4orf1 peptide fragment comprises SEQ ID NO: 1 or a sequence having at least 65% sequence identity with SEQ ID NO:

1.

22. The method of claim 11, wherein the E4orf1 peptide fragment comprises SEQ ID NO:

1.

23. The method of claim 11, wherein the E4orf1 peptide fragment comprises SEQ ID NO: 2 or a sequence having at least 65% sequence identity with SEQ ID NO:

2.

24. The method of claim 11, wherein the E4orf1 peptide fragment comprises SEQ ID NO: 2.

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