Ad36e4orf1 peptide fragments as Anti-diabetic agents

Abstract

Embodiments of the present disclosure pertain to a composition that includes at least one early gene 4 open reading frame 1 (E4orfl) peptide fragment. The E4orfl peptide fragment includes, without limitation, SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1; SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2; or combinations thereof. Additional embodiments of the present disclosure pertain to methods of modulating cellular glucose uptake by associating cells with a composition of the present disclosure. Further embodiments of the present disclosure pertain to methods of treating or preventing a metabolic disorder in a subject by administering a composition of the present disclosure to the subject.

Description

TITLEAd36E4orfl Peptide Fragments as Anti-diabetic AgentsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 543,338, filed on October 10, 2023. The entirety of the aforementioned application is incorporated herein by reference.SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF13368.P059WO.xml”. The date of the creation of the Sequence Listing is October 10, 2024. The size of the Sequence Listing is 6,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing.BACKGROUND

[0003] A need exists for more effective agents for treating or preventing metabolic disorders. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to a composition that includes at least one early gene 4 open reading frame 1 (E4orfl) peptide fragment. In some embodiments, the E4orfl peptide fragment includes, without limitation, REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence with at least 65% sequence identity to SEQ ID NO: 1 ; YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERV IFPSVRIATLV (SEQ ID NO: 2) or a sequence with at least 65% sequence identity to SEQ ID NO: 2; or combinations thereof.

[0005] Additional embodiments of the present disclosure pertain to methods of modulating cellular glucose uptake by associating cells with a composition of the present disclosure. Further embodiments of the present disclosure pertain to methods of treating or preventing a metabolic disorder in a subject by administering a composition of the present disclosure to the subject.

[0006] The methods of the present disclosure may be utilized to treat or prevent various metabolic disorders in subjects. For instance, in some embodiments, the metabolic disorders include, without limitation, 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 steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or combinations thereof.DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A illustrates how the early gene 4 open reading frame 1 (E4orfl) bypasses the proximal insulin signaling pathway but activates distal insulin signaling.

[0008] FIG. IB shows the E4orfl amino acid sequence, with the PDZ domain binding (PBM) motif underlined.

[0009] FIGS. 2A-2C show nanoparticle transmission electron microscopy (TEM) imaging of void nanoparticles (FIG. 2A), nanoparticles treated with a 25 amino acid E4orfl peptide fragment (FIG. 2B), and nanoparticles treated with E4orfl (FIG. 2C).

[0010] FIG. 3 shows a Western blot of phospho (p)-Akt (~ 60 kDa) and total Akt (~ 60 kDa) expression in 3T3L1 cells treated with void nano-liposomes (Void NP), E4orfl nano-liposomes (E4 NP) and 100 nM insulin (Insulin) for 24 h. Bar graphs show p-Akt levels normalized to total Akt, which is significantly higher in the E4 NP group than void NP groups (ANOVA, P <0.05).

[0011] FIGS. 4A-4B show Western blots of phospho (p)-Akt (~ 60 kDa) and total Akt (~ 60 kDa) expression in 3T3-L1 cells treated with 70 pl of void NP, E4orfl NP, P5 NP, P5L NP, P25 NP and 100 nM insulin (Insulin) for 12 h and 24 hr. Bar graphs show p-Akt levels normalized to total Akt levels, which is not significant in peptide NP treated groups (P5, P5L, P25) compared to void NP treated groups. However, Akt activation is significantly higher in E4 NP group compared to void NP groups and peptides NP treated group (ANOVA, P <0.05).

[0012] FIGS. 5A-5B show Western blots of phospho (p)-Akt (~ 60 kDa) and total Akt (~ 60 kDa) expression in 3T3L1 cells treated with 70 pl of void NP, E4orfl NP, P25 NP and 100 nM insulin (Insulin) for 48 h and 72 hr. Bar graphs show p-Akt levels normalized to total Akt levels, which is not significant in P25 NP treated groups and E4orfl NP compared to void NP treated groups (ANOVA, P <0.05).

[0013] FIGS. 6A-6B show Western blots of phospho (p)-Akt (~ 60 kDa) and total Akt (~ 60 kDa) expression in 3T3-L1 cells treated with 70 pl of void NP, P5 NP, and P25 NP for 12 hr. FIG. 6A shows bar graphs of p-Akt levels normalized to total Akt levels, which is significant in the P25 NP treated group when compared to the void NP treated group (ANOVA, P <0.05). FIG. 6B shows subsequent experiments, which did not show any significant difference between the P25 NP treated group when compared to Void NP treated group.

[0014] FIGS. 7A-7B show P25 NP-mediated glucose uptake results. FIG. 7A shows raw data before normalization, which shows a significant difference in the P25 treated group when compared to the Void NP treated group after 12 hours (ANOVA, P <0.05). FIG. 7B provides additional data, which shows no significant difference in the P25 NP treated group when compared to the Void NP treated group 12 hours after normalization (ANOVA, P <0.05).

[0015] FIGS. 8A-8B show Western blots of phospho (p)-Akt (~ 60 kDa) and total Akt (~ 60 kDa) expression in 3T3-L1 cells treated with 0.208 pM P70 Ad36E4orfl peptide for 12 h and 24 hr. Bar graphs show p-Akt levels normalized to total Akt levels, which is significant in peptide P70- NP treated cells for 12h compared with void NP treated cells.DETAILED DESCRIPTION[00161 It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0017] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0018] Metabolic disorders represent a significant public health issue. For instance, diabetes mellitus is one of the most common complex metabolic disorders with heterogenous pathogenesis. Type 1 Diabetes (T1D) is characterized by defective insulin secretion by pancreatic beta-cells and Type 2 Diabetes (T2D) reflects the impairment in insulin-sensitive tissues to respond to insulin.

[0019] An estimated 9.3% (463 million people) of the world’s population was diagnosed with diabetes by year 2019, which is expected to rise to 10.2% (578 million) by 2030 and 12.4% (700 million) by 2045. According to the National Diabetes Statistics Report 2022, more than 130 million people of all ages have been living with diabetes or prediabetes in the United States. Among them, more than 90% of people have T2D. Therefore, diabetes with its ever-increasing alarming prevalence has emerged as a challenging health problem worldwide.

[0020] Insulin resistance is one of the common features of T2D. As illustrated in FIG. 1A, the insulin signaling pathway mainly consists of a metabolic and a mitogen arm. Most metabolic effects of insulin are related to the PI3K / AKT pathway. Therefore, the pathogeneses due to insulin resistance are mainly connected with the PI3K / AKT pathway. Conventionally, the PI3K / AKT pathway is divided into proximal and distal insulin signaling. 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) that subsequently activates the PI3K / AKT pathway.

[0021] Phosphorylation of the PI3K / AKT pathway activates Akt, leading to glucose transporter 4 (Glut 4) translocation to the cell membrane for glucose uptake. Conformational changes of IRS1 and IRS2 refer to the proximal signaling pathway while the PI3K / AKT and Glut 4 indicate the distal signaling pathway. However, the proximal signaling pathway becomes incapable of sending signals for cellular uptake of glucose from the blood during insulin resistance status. Moreover, most of the available anti-diabetic medications such as secretagogues, insulin mimetics, or sensitizers focus on the activation of the proximal insulin signaling for enhancing cellular glucose uptake.

[0022] As such, a need exists for more effective agents for treating or preventing metabolic disorders. In particular, a need exists for more effective agents that can activate distal insulin signaling pathways in insulin resistance or in the absence of insulin. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0023] Compositions

[0024] In some embodiments, the present disclosure pertains to a composition that includes at least one early gene 4 open reading frame 1 (E4orfl) peptide fragment. In some embodiments, the E4orfl peptide fragment includes, without limitation, REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence with at least 65% sequence identity to SEQ ID NO: 1;YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERV IFPSVRIATLV (SEQ ID NO: 2) or a sequence with at least 65% sequence identity to SEQ ID NO: 2; or combinations thereof. As set forth in more detail herein, the compositions of the present disclosure can have numerous embodiments.

[0025] E4orf 1 peptide fragments

[0026] The compositions of the present disclosure can include various E4orfl peptide fragments. For instance, in some embodiments, the E4orf 1 peptide fragment includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 75% sequence identity to SEQ ID NO: 1 . In some embodiments, the E4orf 1 peptide fragment includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the E4orfl peptide fragment includes SEQ ID NO: 1.

[0027] In some embodiments, the E4orfl peptide fragment includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes a sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the E4orfl peptide fragment includes SEQ ID NO: 2.

[0028] Delivery agents

[0029] In some embodiments, the E4orfl peptide fragments of the present disclosure may be associated with a delivery agent. The E4orfl peptide fragments of the present disclosure may be associated with various delivery agents. For instance, in some embodiments, the delivery agent includes, without limitation, a particle, a nanoparticle, a lipid-based nanoparticle, a liposome, a nanoliposome, or combinations thereof.

[0030] The E4orfl peptide fragments of the present disclosure may be associated with delivery agents in various manners. For instance, in some embodiments, the E4orfl peptide fragment is encapsulated in the delivery agent. In some embodiments, the E4orfl peptide fragment is on a surface of the delivery agent. In some embodiments, the E4orfl peptide fragment is non- covalently linked to the delivery agent.

[0031] Composition components and forms

[0032] The compositions of the present disclosure may include various additional components. For instance, in some embodiments, the compositions of the present disclosure include at least one solubilizing agent. In some embodiments, the solubilizing agent includes, without limitation, polyethylene glycol, glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylated glycerides, polyoxyethylated oleic glycerides, polysorbates, sorbitan monooleate, hydroxypropyl-beta- cyclodextrin (HPCD), polyoxyl 40 hydrogenated castor oil, polyoxyl hydroxystearates, or combinations thereof.

[0033] The compositions of the present disclosure may be in various forms. For instance, in some embodiments, the compositions of the present disclosure may be in the form of a liquid. In some embodiments, the compositions of the present disclosure may be in the form of a solid. In some embodiments, the compositions of the present disclosure may be in lyophilized form.

[0034] Uses

[0035] The compositions of the present disclosure may have various uses. For instance, in some embodiments, the compositions of the present disclosure are suitable for use in treating or preventing a metabolic disorder in a subject. In some embodiments, the metabolic disorder includes, without limitation, prediabetes, Type 1 Diabetes (T1D), Type 2 Diabetes (T2D), insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or combinations thereof.

[0036] Methods of modulating cellular glucose uptake

[0037] Additional embodiments of the present disclosure pertain to methods of modulating cellular glucose uptake by associating cells with a composition of the present disclosure. The compositions of the present disclosure may be associated with various cells. For instance, in some embodiments, the cells include fat cells, muscle cells, or liver cells.

[0038] The compositions of the present disclosure may have various effects on cells. For instance, in some embodiments, the compositions of the present disclosure increase cellular glucose uptake. In some embodiments, the compositions of the present disclosure increase cellular glucose uptake independently of proximal insulin signaling pathways. In some embodiments, the compositions of the present disclosure can cause an increase in cellular glucose uptake in fat cells, muscle cells, or liver cells.

[0039] The compositions of the present disclosure may become associated with cells in various manners. For instance, in some embodiments, the association of cells with the compositions of the present disclosure occurs in vitro. In some embodiments, the association of cells with the compositions of the present disclosure occurs in vivo in a subject.

[0040] Methods of treating or preventing metabolic disorders

[0041] Additional embodiments of the present disclosure pertain to methods of treating or preventing a metabolic disorder in a subject by administering a composition of the present disclosure to a subject. As set forth in more detail herein, the methods of the present disclosure can have numerous embodiments.

[0042] Metabolic disorders

[0043] The methods of the present disclosure may be utilized to treat or prevent various metabolic disorders in subjects. For instance, in some embodiments, the metabolic disorders include, without limitation, 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 steato-hepatitis (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).

[0044] Without being bound by theory, the compositions of the present disclosure may treat or prevent metabolic disorders in various manners. For instance, in some embodiments, the compositions of the present disclosure reduce endogenous insulin levels in the blood. In some embodiments, the compositions of the present disclosure improve blood glucose levels by reducing high levels of glucose in the blood, reducing the rise in glucose levels or the duration of glucose elevation expected after eating food, or reducing glucose output from the liver. In some embodiments, the compositions of the present disclosure prevent an increase in blood insulin levels.

[0045] In some embodiments, the compositions of the present disclosure increase cellular glucose uptake in a subject. In some embodiments, the compositions of the present disclosure increase cellular glucose uptake independently of proximal insulin signaling pathways. In some embodiments, the compositions of the present disclosure can cause an increase in cellular’ glucose uptake in fat cells, muscle cells, liver cells, or combinations thereof.

[0046] Administration

[0047] The compositions of the present disclosure may be administered to subjects in various manners. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, electroporation, transfection, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, injection, intrathecal injection, intra-articular administration, topical administration, central administration, peripheral administration, aerosol-based administration, nasal administration, intranasal administration, transmucosal administration, transdermal administration, parenteral administration, or combinations thereof. In some embodiments, the administration occurs by a method that includes, without limitation, oral administration, intramuscular administration, intranasal administration, subcutaneous administration, transdermal administration, intravenous administration, or combinations thereof.

[0048] Subjects

[0049] The compositions of the present disclosure may be administered to various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal includes, without limitation, a horse, a rabbit, a mouse, a rat, a pig, a sheep, a cow, a dog, or a cat. In some embodiments, the non-human mammal is a domestic animal, such as a dog or a cat. In some embodiments, the subject is suffering from a metabolic disorder. In some embodiments, the subject is vulnerable to a metabolic disorder.

[0050] Additional Embodiments

[0051] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0052] Example 1. Ad36E4orfl peptides as an anti-diabetic therapeutic

[0053] In prior studies, Applicant identified a 125 amino acid protein encoded by the E4orfl gene (i.e., early gene 4 open reading frame 1) from a human adenovirus (Ad36) that shows anti- hyperglycemic effects in vitro (preadipocytes, adipocytes, myoblast cells, and / or skeletal muscles) and in animal models. This protein (Ad36E4orfl) shows potential in reducing blood glucose levels and increasing cellular glucose uptake, despite insulin resistance, by bypassing the impaired proximal insulin signaling pathway and preferentially activating the distal insulin signaling pathway.

[0054] Therefore, the insulin-independent action of Ad36E4orfl makes it a novel and potential drug for both type 1 and type 2 diabetes disorders. In addition, this protein shows in vivo and in vitro suppression of glucose output and lipid accumulation in the liver, which indicates the promising role of Ad36E4orfl in preventing progression of hepatic steatosis. Hence, this protein offers a research opportunity to develop a new anti-diabetic drug with multiple potential advantages.

[0055] The Ad36E4orfl protein is not naturally expressed in the human body. Therefore, this protein should preferably associate with a delivery system to enter cells and exert its action. Applicant’s lab previously established a lipid-based nanoparticle, which shows successful transport and anti-diabetic effects of the E4orfl protein through distal cell signaling pathway in mouse-derived preadipocyte (3T3-L1) cell lines. However, the purified E4orfl protein contains a Glutathione Sepharose Transferase (GST) tag, which is commonly used for protein purification. Removal of this tag is challenging as it compromises total protein yield. Additionally, using a tagged protein could elicit an immune response in vivo (e.g., opsonization by serum proteins and clearance by the mononuclear phagocytic system), which is not desirable.

[0056] Therefore, Applicant aimed in this Example to establish peptides of smaller amino acid lengths based on the Ad36E4orfl protein sequence as an alternative approach. The Ad36E4orfl protein sequence consists of a PDZ domain binding (PBM) motif, which is required for its function (FIG. IB). In fact, previous experiments by Applicant showed that Ad36E4orfl needs the PBM region to exert its function. Hence, Applicant designed peptides of varying amino acid lengths containing the PBM sequence.

[0057] Example 1,1. Synthesis of E4orf 1 peptide fragments with PBM domains

[0058] Applicant’s main objective was to determine if nanoparticle-mediated in vitro and in vivo delivery of E4orfl peptide fragments mimics whole Ad36E4orfl protein action. Peptide-based agents rather than recombinant proteins have some advantages, such as more site selectivity, more specificity, less immunogenicity, and higher therapeutic effects. Applicant tested peptides of varying numbers of amino acids in length (5, 10 and 25 amino acids sequence) of the E4orfl protein.

[0059] Table 1 summarizes the different E4orfl peptide fragments tested in this Example.E4orfl peptide fragment name Sequence (NH2 to COOH)P5 (SEQ ID NO: 4) IATLVP5L (SEQ ID NO: 5) KKKK-IATLVP25 (SEQ ID NO: 1) REGHPVGTLLLERVIFPSVRIATLVTable 1. The E4orfl peptide fragments tested (the PBM domain is underlined).

[0060] As shown in Table 1, the P5 peptide fragment has five amino acid sequences. The molecular weight of this peptide is 515.35 Dalton. The P5L peptide shares the same sequence as the P5 peptide along with four additional lysine residues. This synthetic peptide is hydrophilic in nature. The molecular weight of this peptide is 1028.3 Dalton. Additionally, the molecular weight of the P25 peptide is 2773.33 Dalton. These peptides of smaller amino acid sequence in length without the GST tag will be a shorter version of the full length E4orf 1 protein, which Applicant expects to be less immunogenic and cost-effective.

[0061] Example 1,2, Characterization of liposomes

[0062] Nanoliposomes are clinically relevant to deliver Ad36E4orfl. Liposomes are among the well-known nanoparticles and liposomal pharmaceutical products that were approved by the US Food and Drug Administration (FDA) in 1995 for the treatment of chemotherapy-refractory acquired immune deficiency syndrome (AIDS)-related Kaposi’s sarcoma. Current applications of liposomes are in immunology, dermatology, vaccine adjuvant, eye disorders, brain targeting, infective disease and in tumor therapy. Liposome-mediated drug delivery systems also inhibit rapid clearance of liposomes by controlling size, charge, and surface hydration. In addition, liposomes protect encapsulated substances from physiological degradation. Furthermore, liposomes provide higher therapeutic effects, optimal biocompatibility, and optimal safety. Therefore, in delivering Ad36E4orfl and its peptide fragments, nanoparticlc technology can protect peptides and proteins from degradation, improve the half-life of peptides and proteins, control protein release, and target different tissues in the body.

[0063] To analyze and confirm the morphology and size of nanoparticles, Applicant performed transmission electron microscopy (TEM) imaging of the nanoparticles. FIGS. 2A-2C show the void nanoparticle (FIG. 2A), nanoparticles treated with peptide-25 (FIG. 2B), and nanoparticles treated with E4orfl protein (FIG. 2C). As seen in FIGS. 2A-2C, the spherical shape of the nanoparticles is preserved when encapsulated with the 25aa peptide and the 125aa Ad36E4orfl protein, which is consistent with Applicant’s previous observations. The size of the nanoparticles extracted from the images were in the range of 70-100 nm. TEM imaging confirms the desired morphology and the size distribution of Applicant’s nanoparticles (FIGS. 2A-2C).

[0064] Example L3. Activation of insulin signaling by Ad36E4orfl

[0065] Applicant examined if the 125aa Ad36E4orfl protein can be delivered by nanoliposome encapsulation to examine Akt phosphorylation as activation of the distal insulin signaling pathway. As illustrated in FIG. 3, Ad36E4orfl nanoliposome (E4orfl-NP) treated 3T3-L1 cells significantly increase pAkt levels compared with the control cells treated with void nanoliposome (void) after 24 h treatment. Positive control insulin treated cells also show significant phosphorylation of Akt (FIG. 3).

[0066] Applicant next examined the ability of 50uM Ad36E4orfl peptides P5L and P25 in improving Akt phosphorylation in 3T3-L1 cells. As illustrated in FIGS. 4A-4B, 12h or 24h treatment with nanoliposome encapsulated P5L or P25 did not increase Akt phosphorylation compared with void treated control cells. Furthermore, 48h or 72h treatment with nanoliposome encapsulated 50uM peptide 25 (P25) did not increase Akt phosphorylation compared with void treated control cells (FIGS. 5A-5B).

[0067] Applicant next determined a dose dependent effect of P5 and P25 (0.25ug / ul and 0.50ug / ul) on Akt phosphorylation in 3T3-L1 cells. As seen in FIGS. 6A-6B, nanoliposome encapsulated 0.25ug / ul P25 showed significant Akt phosphorylation following 12h treatment compared with void treated control cells (FIG. 6A). However, a follow up experiment did not show the previously observed result (FIG. 6B).

[0068] Example 1.4. Glucose uptake assay

[0069] Applicant next performed the glucose uptake assay to determine the ability of Ad36E4orfl P25 to enhance cellular glucose uptake. Murine 3T3-L1 cells were treated with nanoliposome encapsulated 0.25ug / ul P25 for 12h and exposed to 3H radiolabeled glucose. Amount of glucose in cell lysates was measured using a scintillation counter. As seen in FIGS. 7A-7B, Ad36E4orfl P25 treated cells show increased cellular glucose compared with void treated control cells (FIG. 7A). However, when these numbers were normalized to amount of protein, there was no difference (FIG. 7B). Collectively, the data show that Ad36E4orf 1 25aa peptide (P25) could improve cellular glucose uptake by activating the insulin independent distal insulin signaling.

[0070] Example 2. Activation of Insulin signaling by a 70aa Ad36E4orfl peptide

[0071] In Example 1, Applicant reported that the Ad36E4orfl 25aa peptide (P25) could improve cellular glucose uptake by activating the insulin independent distal insulin signaling. To further examine other Ad36E4orfl peptides, Applicant tested the effect of a peptide 70aa in length as shown in Table 2.E4orfl peptide fragment name Sequence (NH2 to COOH)P70 (SEQ ID NO: 2) YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTLLLERVIFPSVRIATLVTable 2. The E4orfl peptide fragment tested in Example 2 (the PBM domain is underlined).

[0072] As shown in Table 2, P70 is a 70 amino acid sequence synthetic peptide. The molecular weight of this peptide is 8083.38 Dalton.

[0073] Murine 3T3-L1 pre-adipocytes were treated with either 0.208 uM P70 Ad36E4orfl peptide encapsulated nanoliposomes (peptide) or void nanoliposomes (void) for 12 or 24 hours (h). As shown in FIG. 8A, 3T3-L1 cells treated with P70 Ad36E4orfl peptide for 12h significantly increase pAkt protein expression compared with void treated control cells. Cells treated for 24h with P70 Ad36E4orfl peptide did not show any increase in pAkt expression (FIG. 8A).

[0074] Next, Applicant examined Ad36E4orfl protein expression in these cells. Protein lysates from transgenic mice expressing the 125aa Ad36E4orfl protein was used as a positive control. P70 Ad36E4orfl peptide treated cells for 12h show protein expression, which is absent in the void cells, confirming protein translation of the peptide (FIG. 8B).

[0075] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the ail without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising at least one early gene 4 open reading frame 1 (E4orfl) peptide fragment, wherein the E4orfl peptide fragment is selected from the group consisting ofREGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence with at least 65% sequence identity to SEQ ID NO: 1;YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTL LLERVIFPSVRIATLV (SEQ ID NO: 2) or a sequence with at least 65% sequence identity to SEQ ID NO: 2; or combinations thereof.

2. The composition of claim 1, wherein the E4orfl peptide fragment is associated with a delivery agent.

3. The composition of claim 2, wherein the delivery agent is selected from the group consisting of a particle, a nanoparticle, a lipid-based nanoparticle, a liposome, a nanoliposome, or combinations thereof.

4. The composition of claim 2, wherein the E4orf 1 peptide fragment is encapsulated in the delivery agent.

5. The composition of claim 1, wherein the E4orfl peptide fragment comprises SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1.

6. The composition of claim 1, wherein the E4orfl peptide fragment comprises SEQ ID NO: 1.

7. The composition of claim 1, wherein the E4orfl peptide fragment comprises SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2.

8. The composition of claim 1, wherein the E4orfl peptide fragment comprises SEQ ID NO: 2.

9. The composition of claim 1, wherein the composition is suitable for use in treating or preventing a metabolic disorder 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 steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or combinations thereof.

11. A method of treating or preventing a metabolic disorder in a subject, said method comprising: administering a composition to the subject, wherein the composition comprises at least one early gene 4 open reading frame 1 (E4orfl) peptide fragment, wherein the E4orfl peptide fragment is selected from the group consisting of:REGHPVGTLLLERVIFPSVRIATLV (SEQ ID NO: 1) or a sequence with at least 65% sequence identity to SEQ ID NO: 1;YQGRFMALNDYHARGILTQSDVIFAGRRHDLSVLLFNHTDRFLYVREGHPVGTL LLERVIFPSVRIATLV (SEQ ID NO: 2) or a sequence with at least 65% sequence identity to SEQ ID NO: 2; or combinations thereof.

12. The method of claim 11, wherein the administration occurs by 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, topical 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 occurs by a method selected from the group consisting of oral administration, intramuscular administration, intranasal administration, subcutaneous administration, transdermal administration, intravenous administration, or combinations 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 steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, or combinations thereof.

15. The method of claim 11, wherein the composition reduces endogenous insulin levels 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 subject is a human being.

18. The method of claim 11, wherein the E4orfl peptide fragment is associated with a delivery agent.

19. The method of claim 18, wherein the delivery agent is selected from the group consisting of a particle, a nanoparticle, a lipid-based nanoparticle, a liposome, a nanoliposome, or combinations thereof.

20. The method of claim 11, wherein the E4orfl fragment is encapsulated in the delivery agent.

21. The method of claim 11, wherein the E4orfl peptide fragment comprises SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1.

22. The method of claim 11, wherein the E4orfl peptide fragment comprises SEQ ID NO: 1.

23. The method of claim 11, wherein the E4orfl peptide fragment comprises SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2.

24. The method of claim 11, wherein the E4orfl peptide fragment comprises SEQ ID NO: 2.

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