CN112168969A - Inhibition effect of DDIT4L and functional small peptide thereof on glioblastoma - Google Patents

Abstract

The inhibitory effect of DDIT4L and its functional small peptides on glioblastoma is disclosed herein. Specifically disclosed is the use of a DNA damage inducing transcript 4-like (DDIT4L) active substance in the manufacture of a product for the prevention and/or treatment of glioma and a symptom associated therewith in a subject, wherein said active substance is selected from the group consisting of: a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof.

Description

Inhibition effect of DDIT4L and functional small peptide thereof on glioblastoma

Technical Field

The present invention belongs to the field of biotechnology and medicine. Specifically, the invention relates to the inhibition effect of DNA damage induced transcript 4-like (DDIT4L) active substances (such as DDIT4L and functional small peptides thereof) on glioma (such as glioblastoma), and related products and applications thereof.

Background

Glioma (glioma) is a tumor derived from neuroepithelium, accounts for 40% -50% of craniocerebral tumors, is the most common primary intracranial tumor, and has an annual incidence rate of 3-8 persons/10 ten thousand of people. The Glioblastoma (GBM) among the gliomas is the most clinically fatal primary brain tumor.

While there are many grading systems for gliomas today, the most common is that set by the World Health Organization (WHO). According to this classification system, brain gliomas are classified into low-grade gliomas (LGG, WHO grade 1-2, well-differentiated gliomas, which are not benign but have a relatively good prognosis in patients) and high-grade gliomas (HGG, WHO grade 3-4, low-differentiated gliomas, which have a high degree of malignancy and a poor prognosis). Among these, the classical cytopathology, so-called anaplastic glioma, corresponds to WHO grade 3; while glioblastoma corresponds to the most malignant WHO grade 4 glioma.

Recent studies in tumor genetics have shown that: the Isocitrate Dehydrogenase (IDH)1/2 gene is frequently mutated in malignant glioma tissues. Clinical data currently indicate that about 80% of gliomas pathologically diagnosed as grade II or III have IDH1/2 gene mutations, and less than 10% of gliomas of grade IV (also known as glioblastoma) have IDH1/2 gene mutations.

The wild-type IDH is a metabolic enzyme which catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, which participates in the tricarboxylic acid cycle to generate ATP by activating ATP synthase. The most common site of IDH1/2 gene mutation in glioma patient tissues is IDH1^R132H. The mutated IDH is a newly mutated metabolic enzyme that catalyzes the oxidative decarboxylation of isocitrate to D-2-hydroxyglutarate. In mutant IDH gliomas, accumulated D-2-hydroxyglutarate can increase RNA N by inhibiting ATP synthase and mTOR activity⁶Methyl adenosine is modified to inhibit tumor growth. These findings have led to new strategies for the treatment of gliomas with IDH mutations.

However, it is not known whether endogenous inhibitors that inhibit energy metabolism of cells and simultaneously mediate apoptosis exist in glioblastoma. Therefore, the finding of molecules which can inhibit ATP synthetase and promote apoptosis has important significance and urgency for inhibiting glioma (especially GBM).

DNA Damage-induced transcript 4-like (DDIT4L) protein is an in vivo stress protein. The DDIT4L molecule was first discovered in screening Drosophila's TOR signaling pathway and negative regulators affecting cell size, and was named "Charybdis" in Drosophila. In mammals, it is named DDIT4L (or REDD2/RTP 801L); it has a homologous protein DDIT4 (or REDD1/RTP801) which is 36% identical in protein sequence to DDIT 4L. Both proteins have been shown to be upstream inhibitors of mTOR in different tissue and cell models.

In human tissues, the DDIT4L gene is mainly expressed in skeletal muscle, while the DDIT4 gene is expressed in various tissues throughout the body. In pathological conditions, hypermethylated DDIT4L was found to be present in melanoma, on the other hand, DDIT4L may also inhibit pathological myocardial hypertrophy and promote autophagy.

However, the association of the DDIT4L gene and/or protein and/or active fragments thereof with gliomas has never been revealed in the art.

Disclosure of Invention

The invention provides the inhibition effect of DDIT4L and functional small peptide thereof on glioblastoma and related products and applications thereof.

In a first aspect of the present invention there is provided the use of a DNA damage inducing transcript 4-like (DDIT4L) active substance in the manufacture of a product for the prevention and/or treatment of glioma and symptoms associated therewith in a subject, wherein said active substance is selected from the group consisting of: a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof.

In some embodiments, the sequence of the DDIT4L polypeptide is selected from:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2; or

(b) A polypeptide that is homologous or has sequence identity (e.g., greater than 80% homologous or greater than 80% sequence identity, e.g., 80%, 85%, 90%, 95%, 98%, 99%) to the amino acid sequence set forth in SEQ ID NO. 2, and that has binding and inhibitory activity against the α subunit of ATP synthase; or

(c) And (b) polypeptide which is derived from (a) or (b) and has ATP synthetase alpha subunit binding and inhibiting activity.

In some embodiments, the nucleic acid molecule encoding a DDIT4L polypeptide (e.g., the DDIT4L polypeptide of (a) - (c)) is selected from the group consisting of:

(i) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO. 1; or

(ii) (ii) a molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (i);

(iii) a nucleic acid molecule that is homologous to or has sequence identity (e.g., greater than 80% homologous or greater than 80% sequence identity, e.g., 80%, 85%, 90%, 95%, 98%, 99%) to the nucleotide sequence set forth in SEQ ID NO:1 and encodes a polypeptide having ATP synthase alpha subunit binding and inhibitory activity;

(iv) (iv) a nucleic acid molecule which is obtained by substituting, deleting or adding one or more nucleotides from the nucleotide sequence of (i) or (ii) or (iii) and which encodes a polypeptide having the binding and inhibitory activity of the alpha subunit of ATP synthase.

In some embodiments, the active fragment is selected from the group consisting of:

(a') a fragment having a peptide portion corresponding to V125-P132 of a DDIT4L polypeptide, such as a polypeptide having an amino acid sequence shown in SEQ ID NO. 4 or 6; or

(b') a polypeptide which is homologous or has sequence identity (e.g., more than 80% homologous or has more than 80% sequence identity, such as 80%, 85%, 90%, 95%, 98%, 99%) to the amino acid sequence represented by SEQ ID NO:4 or 6 and which has ATP synthase alpha subunit binding and inhibitory activity; or

(c ') a polypeptide derived from (a) or (b) having an ATP synthase alpha subunit binding and inhibitory activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence of (a ') or (b '); and/or

The nucleic acid molecule encoding an active fragment of a DDIT40L polypeptide (e.g., the active fragments of (a ') through (c')) is selected from the group consisting of:

(i') the nucleic acid molecule corresponding to the active fragment contained in SEQ ID NO: 1; or

(ii ') a molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (i');

(iii') a nucleic acid molecule which is homologous or has sequence identity (e.g., greater than 80% homologous or has greater than 80% sequence identity, e.g., 80%, 85%, 90%, 95%, 98%, 99%) to the nucleotide sequence set forth in SEQ ID NO:3 and which encodes a polypeptide having ATP synthase alpha subunit binding and inhibitory activity;

(iv ') a nucleic acid molecule which is obtained by substituting, deleting or adding one or more nucleotides from the nucleotide sequence of (i') or (ii ') or (iii') and which encodes a polypeptide having an ATP synthase alpha subunit binding and inhibitory activity.

In some embodiments, the promoter is selected from the group consisting of: a substance that increases the level of or facilitates the function of DDIT4L polypeptide or an active fragment thereof, such as an overexpression vector for DDIT4L or an active fragment thereof or a coding sequence thereof; an exogenous DDIT4L polypeptide or active fragment; naked DNA of the coding sequence of DDIT 4L; liposome-encapsulated DNA of the DDIT4L coding sequence; a precursor protein or conjugate or complex capable of being converted in vivo to DDIT4L or an active fragment thereof.

In some embodiments, the polypeptide or active fragment thereof is linked to a carrier, half-life extender, signal peptide, or the like, for example to the cell-penetrating peptide TAT. In some embodiments, the linker is DDIT4L or an active fragment thereof linked to a TAT peptide, such as a TAT DDIT4L V125-P132 small peptide.

In some embodiments, the glioma is a WHO grade 1 glioma, a WHO grade 2 glioma, a WHO grade 3 glioma, or a WHO grade 4 glioma.

In some embodiments, the glioma is a glioblastoma.

In some embodiments, the glioma is a glioma with wild-type IDH (e.g., a glioblastoma) or a glioma with an IDH1/2 mutation (e.g., IDH 1)^R132HGliomas).

In some embodiments, the glioma is a proneural (Pro), neurogenic (neural), classical (classical) or mesenchymal (Mes) glioma.

In some embodiments, the glioma is a supratentorial glioma, a subtopical glioma, or a pontocerebral glioma.

In some embodiments, the glioma-associated condition is selected from the group consisting of glioma-induced: headache, nausea, vomiting, epilepsy, blurred vision, vision loss, pain in the limbs, numbness, weakness in muscle strength, movement and sensation disorders, speech expression and difficulty in understanding.

In some embodiments, the preventing and/or treating comprises: preventing or delaying the onset and/or development of glioma, promoting the regression or disappearance of glioma, alleviating symptoms of glioma (such as those described above), and/or prolonging survival.

In some embodiments, the DDIT4L active performs one or more of the following functions: bind to and inhibit ATP synthase alpha subunit; inhibition of mitochondrial metabolic function; increase mitochondrial membrane permeability; promote release of cytochrome C and/or caspases (e.g., caspase 3) in mitochondria; inducing apoptosis of tumor cells (e.g., glioma cells); inhibition of the mTOR signaling pathway; inhibiting tumor cell growth, proliferation and/or development.

In some embodiments, the subject is a human or non-human mammal, such as a chimpanzee, ape, monkey, horse, cow, sheep, dog, cat, or mouse.

In some embodiments, the product is a pharmaceutical composition or kit, e.g., in a form suitable for administration by a formulation selected from the group consisting of: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, intracranial administration, and intratumoral administration.

In some embodiments, the product further comprises other substances for preventing and/or treating glioma or symptoms associated with glioma, such as one or more selected from the group consisting of: temozolomide, bevacizumab avastin; alternatively, the product is used in combination with surgery, radiation therapy and/or chemotherapy.

In some aspects of the present application, there is provided a product for preventing and/or treating glioma and symptoms associated therewith comprising:

(A) a therapeutically or prophylactically effective amount of a DNA damage inducing transcript 4-like (DDIT4L) polypeptide, an active fragment thereof, a nucleic acid molecule encoding said polypeptide or fragment, or a promoter thereof, e.g., a polypeptide, an active fragment thereof, a nucleic acid molecule encoding said polypeptide or fragment, or a promoter as defined in claim 2;

(B) a pharmaceutically acceptable carrier or excipient;

(C) optionally, one or more other active substances for the prophylaxis and/or treatment of gliomas and their associated symptoms,

for example, the product may be characterized as described herein (e.g., above).

In some embodiments, the product is a pharmaceutical composition or kit, e.g., in a form suitable for administration by a formulation selected from the group consisting of: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, intracranial administration, and intratumoral administration.

In some embodiments, the product further comprises the additional active selected from one or more of the following group: temozolomide, bevacizumab avastin; alternatively, the product is used in combination with surgery, radiation therapy and/or chemotherapy.

In some aspects of the present application, there is provided the use of a DNA damage inducing transcript 4-like (DDIT4L) active substance in the manufacture of a product for inhibiting mitochondrial metabolic activity and/or treating a disease or condition associated with increased mitochondrial metabolic activity, wherein the active substance is selected from the group consisting of: a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof.

In some embodiments, the active and/or product is as described herein (e.g., supra).

In some embodiments, the disease or condition associated with overactive mitochondrial metabolism is selected from: tumorigenesis, tumor migration.

In other aspects of the present application, there is provided a method of preventing and/or treating glioma and its associated symptoms in a subject, the method comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a DNA damage inducing transcript 4-like (DDIT4L) active and/or a product of the present application, wherein the active is selected from the group consisting of: a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof.

In some embodiments, various features in the methods may be as described herein (e.g., supra).

Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the spirit and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will now be further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the invention only and not for the purpose of limiting the scope of the invention.

FIG. 1: the expression of DDIT4L in gliomas is positively correlated with the malignancy of the gliomas:

a: analyzing up-regulated genes in glioma in different classification modes by using a TCGA database;

b: analyzing the expression levels of different genes in GBM and LGG using a TCGA database;

c: photograph of immunohistochemical staining (DAB staining) of DDIT4L expressed in GBM (left panel: hGBM 21359; right panel: hLGG 14023);

d to e: immunofluorescence double labeling and cell flow assay DDIT4L mapped to GFAP, oligo2, Nestin, and CD 133.

FIG. 2: DDIT4L inhibited glioma cell proliferation:

a: expression of DDIT4L in GBM patient tissue;

b to c: immunohistochemistry and cell flow assay for the localization of DDIT4L to hypoxia inducible factor 1 α (HIF1 α) and vascular endothelial cell marker CD 31;

d: effect of overexpression of DDIT4L in primary cells (hGBM11879) on neoplastic cell outgrowth.

FIG. 3: DDIT4L binds to the alpha subunit of ATP synthase:

a: co-immunoprecipitation of GBM of DDIT4L and myc of U87MG cells transfected with myc and His-tag;

b: immunostaining detects the localization of DDIT4L and ATP synthase alpha subunit in GBM tissue;

c: co-immunoprecipitation in HEK293T cells co-transfected with DDIT4L and the ATP synthase alpha or beta subunit;

d: GST pull-down experiment detects the combination of ATP synthetase alpha subunit and DDIT 4L;

e: binding of the purified ATP synthase alpha subunit to the DDIT4L protein;

f: co-immunoprecipitation assay the site of DDIT4L that binds to the ATP synthase alpha subunit, where DDIT4L Δ 1, DDIT4L Δ 2, and DDIT4L Δ 3 correspond to DDIT4L lacking S96-C106, V125-P132, and K141-R166, respectively;

g: GST pull-down experiments examine the binding regions on the α subunits of ATP synthase, where ATP5A Δ 1. DELTA.5 corresponds to the α subunits of ATP synthase lacking V118-V131, K132-I158, I179-G192, L471-A486, and G491-I504, respectively;

h: the binding sites of DDIT4L and the alpha subunit of ATP synthetase are analyzed by homologous protein structure modeling and in-silicon docking model.

FIG. 4: DDIT4L inhibits ATP synthase activity and mediates glioma cell apoptosis:

a: effect of transfection of DDIT4L on cellular ATP synthesis;

b: effect of transfection DDIT4L on mitochondrial membrane potential hyperpolarization;

c: effect of transfection of DDIT4L on tumor cell basal oxygen consumption rate and ATP synthase-induced oxygen consumption rate;

d: immunostaining detects the effect of DDIT4L transfection on mitochondrial fusion, and Fragmented, intervening and tubular in the figure represent small fragments, intermediate and long tubular, respectively;

e: observing the influence of the transfection DDIT4L on mitochondrial fusion and the fracture and regression of the inner and outer membranes of mitochondria by an electron microscope;

f: effect of transfection of DDIT4L on cytochrome c and activated caspase 3 levels in mitochondria and cytoplasm in U87MG cells; tom20 is a protein specifically localized to mitochondria, and high expression thereof indicates successful isolation of mitochondrial components;

g: TUNEL staining to detect the induction of glioma apoptosis by DDIT 4L;

h: effect of transfection of DDIT4L on phosphorylation of S6K.

FIG. 5: DDIT4L and TAT-DDIT4L^V125-P132And (3) inhibiting growth of glioma:

a to c: effect of transfection of DDIT4L or intravenous administration of DDIT4L protein on tumor growth and survival time in human GBM tumor orthotopic xenograft immunodeficient mouse model;

d：TAT-DDIT4L^V125-P132and TAT-DDIT4L^K141-R166Effect on growth of U87MG tumor cells;

e：TAT-DDIT4L^V125-P132effect on ATP synthase-induced oxygen consumption rate of U87MG tumor cells;

f～g：TAT-DDIT4L^V125-P132effect on tumor growth and mouse survival time in human GBM tumor orthotopic xenograft immunodeficiency mouse models.

In the above figures, denotes a P value < 0.05; denotes P value < 0.01; denotes P value < 0.001.

Detailed Description

Prior to this application, it was unknown whether there was an endogenous inhibitor in Glioblastoma (GBM) that had the ability to inhibit energy metabolism of the cell while mediating apoptosis in itself. Endogenous inhibitors against mitochondrial function are presumed to be present in gliomas (especially GBMs) without mutations in IDH. Through the relevant research of the application, the following results are found: in vivo stress protein DNA damage induces transcript 4-like (DDIT4L) proteins that can inhibit glioma growth and development by antagonizing mitochondrial function while initiating apoptosis.

Specifically, it is well known that ATP synthase on mitochondria synthesizes almost all ATP required by cells under aerobic conditions. By analyzing the protein structure of ATP synthetase, the following findings are obtained: the ATP synthase comprises F₁And F₀Two parts. Wherein F₁Contains alpha and beta subunits, and has the main function of catalyzing and synthesizing ATP; and F₀A hydrogen ion channel may be formed.

Previous studies have shown inhibitors of ATP synthase: atpase inhibitor 1(IF1), can bind to the β subunit to inhibit ATP production, but IF1 enhances glioma cell migration and invasion by activating the NF- κ B pathway. In mutant IDH gliomas, D-2-hydroxyglutarate can also bind to the beta subunit to antagonize ATP synthase activity and inhibit tumor growth. In this study, DDIT4L could directly bind to the alpha subunit to antagonize ATP synthase activity and induce apoptosis, thereby inhibiting glioma growth.

In this study, it was found that DNA damage-induced transcript 4-like (DDIT4L) protein is an inhibitor of the growth of endogenous GBM tumor bodies, and can directly bind to the alpha subunit of ATP synthase and inhibit the metabolic function of mitochondria. The DDIT4L gene is highly expressed in the tissues of glioblastoma patients. DDIT4L protein can obviously inhibit the proliferation of GBM cells. Through co-immunoprecipitation, protein mass spectrometry and functional analysis, DDIT4L can directly bind to the alpha subunit of ATP synthase, thereby inhibiting ATP synthase activity and allowing cytochrome C in mitochondria to be released into the cytoplasm, eventually leading to the initiation of apoptosis. Further, the functional fragment of DDIT4L is finely screened, and DDIT4L is found^V125-P132Can obviously inhibit the growth of glioma. Thus, DDIT4L and DDIT4L^V125-P132Can inhibit mitochondrial activity by antagonizing mitochondrial activity to initiate apoptosisGrowth of glioblastoma.

GBM, the most lethal primary brain tumor, is characterized histologically primarily by the presence of numerous hypoxic necrotic regions in tumor body tissue. There are studies that indicate that hypoxic conditions can induce the expression of DDIT4L gene. In this study, we found that there was a significant increase in the expression of DDIT4L in GBM tissue compared to LGG. We speculate that GBM tissue induces increased expression of DDIT4L due to local hypoxia caused by massive proliferation of tumor cells; DDIT4L inhibits tumor growth by inhibiting mitochondrial function and initiating apoptosis. Therefore, changes in the morphology of mitochondria in clinical glioblastomas are prone to mitophagy and apoptosis, leading to local tissue necrosis. When exogenous DDIT4L is administered to tumor cells, DDIT4L binds to ATP5A on the mitochondrial membrane, inhibiting ATP synthase activity. In this process, DDIT4L binds to CYPD on the inner mitochondrial membrane, thereby increasing the permeability of the mitochondrial permeability transition pore (mPTP), causing cytochrome C to be released from the mitochondria into the cytoplasm, thereby activating the performer caspase (caspase) of programmed cell death. On the other hand, DDIT4L binds to the 14-3-3 family protein, inhibits mTOR signaling, inhibits cell proliferation, and induces autophagy. Thus, exogenous DDIT4L or peptide DDIT4L^V125-P132After the addition of GBM cells or tumors, significant apoptosis and tumor suppression occurred. Thus, DDIT4L or peptide DDIT4L^V125-P132Can become a new treatment method of GBM.

It is well known that DDIT4L and DDIT4 have been shown to be inhibitors of the mTOR signaling pathway. The research of DDIT4 proves that it can inhibit mTOR to regulate tumor cell autophagy to resist chemotherapy drug or cell apoptosis caused by anoxic environment, while the research aiming at DDIT4L mainly finds that it inhibits mTOR to regulate the pathological damage process of myocardial cells in myocardial damage research. The above studies suggest that DDIT4L acts contrary to the findings of the present application. Studies have reported that the expression of DDIT4 is up-regulated in tumor tissues of acute myelogenous leukemia, breast cancer, glioblastoma, colon cancer, skin cancer, and lung cancer, suggesting a poor prognosis. The mechanism of DDIT4 for regulating tumor growth is mainly through DDIT4 binding to 14-3-3 protein in cytoplasm, so that the complex formed by the tumor suppressor protein (TSC2) of nodular sclerosis and the 14-3-3 protein is dissociated, and the free TSC2 can inhibit mTORC1, thereby forming an inhibiting effect on the growth of tumor cells.

In some pathological states, molecular regulatory mechanisms similar to those of DDIT4 exist in DDIT4L, but its role in tumor growth is currently being studied only rarely. In this study, it was found that DDIT4L could significantly inhibit the growth of IDH wild-type GBM cells. It was also confirmed that DDIT4L induces apoptosis by inhibiting the activity of ATP synthase on mitochondria. The molecular mechanism of the regulation is mainly that DDIT4L binds ATP5A so as to mediate the regulation effect on ATP synthetase. The amino acid sequence of DDIT4L that binds ATP5A is not present in the protein sequence of DDIT4, nor does it correspond to the sequence of DDIT4 and DDIT4L that binds 14-3-3 protein (Arg-X-X-X-Ser/Thr-X-Pro). Thus, the binding of DDIT4L to ATP5A is a specific effect of DDIT 4L.

Predicted by a protein structure binding model and confirmed by experiments: the V125-P132 small peptide on DDIT4L can be completely embedded into the groove formed by K132-I158 on ATP 5A. After this binding has occurred, the rotational efficiency of the F1 portion of the ATP synthase is significantly reduced, resulting in a significant reduction in ATP synthesis. Thus, increasing the amount of protein of DDIT4L results in decreased ATP synthase activity, and DDIT4L simultaneously binds to CYPD (an important constituent protein of mPTP) on the mitochondrial membrane, resulting in the opening of mPTP, and cytochrome C is released from the mitochondria into the cytosol to initiate the apoptotic program of caspases resulting in apoptosis.

In summary, DDIT4L is an endogenous GBM inhibitor acting on mitochondrial ATP synthase; but also gives a new view to understanding the relationship between mitochondrial function and apoptosis. Furthermore, we identified the polypeptide DDIT4L^V125-P132The function of DDIT4L can be simulated, which provides a good direction for developing GBM treatment drugs.

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

DDIT4L active substance

As used herein, the term "DDIT 4L active substance" or "DNA damage-inducing transcript 4-like active substance" or "active substance of the present application" refers to a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof, and the like, that can inhibit mitochondrial metabolism by binding to a specific region of the ATP synthase alpha subunit, and/or that exert tumor (e.g., glioma, such as glioblastoma) inhibitory effects on DDIT 4L-related substances.

As used herein, the terms "DDIT 4L polypeptide" or "DDIT 4L protein" are used interchangeably to refer to DNA damage-inducing transcript 4-like protein that is an inhibitor of endogenous glioma (especially GBM) by acting on mitochondrial ATP synthase. The DDIT4L protein herein may be selected from: (a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2; or (b) a polypeptide that is homologous or has sequence identity (e.g., greater than 80% homologous or has greater than 80% sequence identity, e.g., 80%, 85%, 90%, 95%, 98%, 99%) to the amino acid sequence set forth in SEQ ID NO. 2, and that has ATP synthase alpha subunit binding and inhibitory activity; or (c) a polypeptide derived from (a) or (b) by substituting, deleting or adding one or more amino acids in the amino acid sequence of (a) or (b), and having binding and inhibitory activity to the α -subunit of ATP synthase.

The proteins or polypeptides herein can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher animal, insect, and mammalian cells). Preferably, the DDIT4L protein or polypeptide is encoded by a human DDIT4L gene or a homologous gene or family gene thereof.

Variants of the DDIT4L protein or polypeptide herein include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein or polypeptide. For another example, the addition of one or more amino acids at the C-terminus and/or N-terminus will not generally alter the function of the protein or polypeptide, e.g., DDIT4L protein or polypeptide may or may not include the initial methionine residue and still be active.

Random mutagenesis may be performed by irradiation or exposure to a mutagenizing agent, or the protein or polypeptide of (b) above may be obtained by site-directed mutagenesis or other known molecular biological techniques.

Depending on the host used in the recombinant production protocol, the protein or polypeptide herein may comprise certain modifications, for example it may be glycosylated or it may be non-glycosylated. The term also includes active fragments and active derivatives of DDIT4L protein.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences that hybridize to the coding sequence of DDIT4L protein under conditions of high or low stringency, and polypeptides or proteins obtained using antisera directed against DDIT4L protein. Other polypeptides, such as fusion proteins comprising DDIT4L protein or fragments thereof, may also be used in the present invention. In addition to the nearly full-length polypeptide, the present invention also includes active or functional fragments of DDIT4L protein.

As used herein, the term "active fragment" or "functional fragment" of a DDIT4L polypeptide or protein refers to the amino acid fragment contained in DDIT4L that has ATP synthase alpha subunit binding and inhibitory activity.

The DDIT4L active fragments herein may be selected from, for example: (a') a fragment having a peptide portion corresponding to V125-P132 of a DDIT4L polypeptide, such as a polypeptide having an amino acid sequence shown in SEQ ID NO. 4 or 6; or (b') a polypeptide which is homologous or has sequence identity (e.g., more than 80% homologous or has more than 80% sequence identity, such as 80%, 85%, 90%, 95%, 98%, 99%) to the amino acid sequence shown in SEQ ID NO 4 or 6 and which has ATP synthase alpha subunit binding and inhibitory activity; or (c ') a polypeptide derived from (a) or (b) by substitution, deletion or addition of one or several amino acids in the amino acid sequence of (a ') or (b ') and having binding and inhibitory activity to the α -subunit of ATP synthase. The active fragment may be obtained by any method known in the art, for example, by artificial synthesis or the like.

For example, an active fragment has at least a V125-P132 segment of DDIT4L protein, or may contain additional amino acid residues opposite in DDIT4L at one or both ends, or may be linked to other moieties (e.g., half-life extenders) that enhance V125-P132 activity (e.g., cell-penetrating peptides, signal peptides) or targeting (e.g., targeting moieties) or prolong its stability and effect in vivo, such as TAT peptide fragments. The active fragment may comprise at least 8, 10, 15, 20, 30 consecutive amino acids up to the full-length DDIT4L protein with the V125-P132 segment as the core segment.

As used herein, the terms "DDIT 4L gene," "DDIT 4L polypeptide coding sequence," or "nucleic acid molecule encoding a DDIT4L polypeptide," all used interchangeably, refer to a sequence encoding a DDIT4L protein or polypeptide described herein, which can be (i) a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 1; or (ii) a molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (i); (iii) a nucleic acid molecule that is homologous to or has sequence identity (e.g., greater than 80% homologous or greater than 80% sequence identity, e.g., 80%, 85%, 90%, 95%, 98%, 99%) to the nucleotide sequence set forth in SEQ ID NO:1 and encodes a polypeptide having ATP synthase alpha subunit binding and inhibitory activity; (iv) (iv) a nucleic acid molecule which is obtained by substituting, deleting or adding one or more nucleotides from the nucleotide sequence of (i) or (ii) or (iii) and which encodes a polypeptide having the binding and inhibitory activity of the alpha subunit of ATP synthase.

As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more. For example, the sequence may be the complement of the sequence defined in (a).

The full-length nucleotide sequence of DDIT4L gene or its fragment can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

It is to be understood that the DDIT4L gene of the invention is preferably obtained from a human, and other genes obtained from other animals that are highly homologous (e.g., have greater than 50%, preferably greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, more preferably greater than 85% such as 85%, 90%, 95%, 98%, or even 99% or greater sequence identity) to the human DDIT4L gene are also within the scope of equivalents contemplated herein. Methods and means for aligning sequence identity are also well known in the art, such as BLAST.

Similarly, active agents herein also include nucleic acid molecules encoding active fragments of DDIT4L herein. The active fragment encoding molecule may be selected from: (i') a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 corresponding to the active fragment; or (ii ') a molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (i'); (iii') a nucleic acid molecule which is homologous or has sequence identity (e.g., greater than 80% homologous or has greater than 80% sequence identity, e.g., 80%, 85%, 90%, 95%, 98%, 99%) to the nucleotide sequence set forth in SEQ ID NO:3 and which encodes a polypeptide having ATP synthase alpha subunit binding and inhibitory activity; (iv ') a nucleic acid molecule which is obtained by substituting, deleting or adding one or more nucleotides from the nucleotide sequence of (i') or (ii ') or (iii') and which encodes a polypeptide having an ATP synthase alpha subunit binding and inhibitory activity. The active fragment can be produced by recombinant expression, or obtained by artificial synthesis.

Also herein are DDIT4L accelerators. The term "enhancer" refers to a substance that increases the level and/or activity of a DDIT4L polypeptide, active fragment, or molecule encoding the same. Accelerators useful herein include, but are not limited to: an expression vector for DDIT4L or an active fragment thereof, exogenous DDIT4L polypeptide or an active fragment thereof, naked DNA of its coding sequence, liposome-encapsulated DNA of its coding sequence, a precursor protein or conjugate or complex capable of being converted in vivo to DDIT4L or an active fragment thereof. In some embodiments, it may be DDIT4L or an active fragment thereof linked to a TAT peptide, such as TAT DDIT4L V125-P132 small peptide.

The DDIT4L actives herein may perform one or more of the following functions: bind to and inhibit ATP synthase alpha subunit; inhibition of mitochondrial metabolic function; promote release of cytochrome C and/or caspases (e.g., caspase 3) in mitochondria; inducing apoptosis of tumor cells (e.g., glioma cells); inhibition of the mTOR signaling pathway; inhibiting tumor cell growth, proliferation and/or development. Thus, the DDIT4L active substance herein has broad application prospects.

Product and application thereof

Also provided herein are products comprising DDIT4L actives and uses thereof. For example, provided herein is a medicament, pharmaceutical composition or kit that may contain an effective amount of the DDIT4L active herein, and a pharmaceutically acceptable carrier.

In some embodiments, the products can be used to prevent or treat glioma and its associated symptoms. For example, the products herein may be used for the prevention and/or treatment of gliomas selected from the group consisting of: WHO grade 1 glioma, WHO grade 2 glioma, WHO grade 3 glioma or WHO grade 4 glioma, such as glioblastoma; gliomas with wild-type IDH or gliomas with IDH1/2 mutation (e.g., IDH 1)^R132HGliomas); neurogenic (Pro), neurogenic (neural), classical (classical) or mesenchymal (Mes) gliomas; supratentorial, infratentorial, or pontocerebral gliomas. In some embodiments, the products herein may be used to alleviate or eliminate a glioma-associated symptom, such as one or more symptoms caused by a glioma selected from the group consisting of: headache, nausea, vomiting, epilepsy, blurred vision, vision loss, pain in the limbs, numbness, weakness in muscle strength, movement and sensation disorders, speech expression and difficulty in understanding. In some embodiments, the preventing and/or treating comprises: preventing or delaying the onset and/or development of glioma, promoting the regression or disappearance of glioma, alleviating symptoms of glioma (such as those described above), and/or prolonging survival.

In some embodiments, the product can be used to prevent or treat a disease or condition associated with mitochondrial hyperfunction. For example, the products herein may be used for the prevention and/or treatment of: tumorigenesis, tumor migration.

As used herein, the terms "comprising" or "including" include "comprising," consisting essentially of … …, "and" consisting of … …. As used herein, the term "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack pub. Co., N.J.1991.

Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances and the like may also be present in these carriers. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

The active substance in the composition accounts for 0.001-99.9 wt% of the total weight of the composition; preferably 1 to 95 wt%, more preferably 5 to 90 wt%, and even more preferably 10 to 80 wt% of the total weight of the composition. The balance of substances such as pharmaceutically acceptable carriers, other additives and the like.

As used herein, the term "unit dosage form" refers to a dosage form that is formulated for single administration of the composition of the present invention for ease of administration, including, but not limited to, various solid dosage forms (e.g., tablets), liquid dosage forms, capsules, sustained release formulations.

In another preferred embodiment of the present invention, the composition is in a unit dosage form or a multi-dosage form, and the content of the active substance therein is 0.01 to 2000 mg/dose, preferably 0.1 to 1500 mg/dose, more preferably 1 to 1000 mg/dose. In another preferred embodiment of the present invention, 1 to 6 doses of the composition of the present invention are applied per day, preferably 1 to 3 doses; most preferably, the dose administered per day is1 dose.

It will be appreciated that the effective dose of the active agent, such as the DDIT4L protein or its coding sequence, used may vary with the severity of the subject to be administered or treated. The specific condition is determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, desired effect), and is within the judgment of a skilled physician.

The composition of the invention can be in solid state (such as granules, tablets, freeze-dried powder, suppositories, capsules and sublingual tablets) or liquid state (such as oral liquid) or other suitable shapes. The administration route can be as follows: (1) direct naked DNA or protein injection; (2) linking cDNA, mRNA and protein of DDIT4L with transferrin/poly L-lysine complex to enhance its biological effect; (3) cDNA, mRNA and protein form a complex with positively charged lipids to overcome the difficulty of crossing cell membranes due to the negative charge of the phosphate backbone; (4) the liposome is used for wrapping cDNA, mRNA and protein and then mediating to enter cells, which is beneficial to the smooth entry of macromolecules and avoids the hydrolysis of various enzymes outside the cells; (5) binding of cDNA, mRNA and protein to cholesterol increased cytoplasmic retention time by 10-fold; (6) the cDNA, mRNA and protein can be specifically transported to target tissues and target cells by using the immunoliposome to transport the cDNA, the mRNA and the protein; (7) transfection of cDNA, mRNA and proteins in vitro into transfected cells (e.g., fibroblasts) also advantageously loads the DDIT 4L-related drug into target cells; (8) electroporation (electroporation), i.e., the introduction of cDNA, mRNA, and proteins into target cells by means of an electric current.

In addition, the products herein may also contain other substances for the prevention and/or treatment of glioma or glioma-associated symptoms, including, but not limited to: temozolomide, bevacizumab avastin, and the like. The various DDIT4L actives (e.g., nucleic acids and peptides) herein may be used in combination with each other, as well as in combination with other drugs and therapeutic treatments (e.g., surgery, radiation therapy, and/or chemotherapy).

Accordingly, also provided herein are methods of preventing and/or treating glioma or a symptom associated therewith, comprising administering to a subject in need thereof a DDIT4L active or product herein. Also provided herein are methods of preventing and/or treating a disease or condition associated with mitochondrial hyperactivity, comprising administering to a subject in need thereof an active substance or product of DDIT4L herein.

Sequence Listing information

The molecules corresponding to the sequence information in the sequence table of the application are as follows:

examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.

The experimental procedures for the conditions not specified in the examples below can be carried out by methods conventional in the art, for example, by referring to the molecular cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989 or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.

Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 expression of DDIT4L in gliomas positively correlated with the malignancy of the gliomas

Materials and methods

Tissue specimen: tumor surgical resection specimens were obtained from Huashan Hospital, affiliated with the university of Zedan, and histologically examined as Glioblastoma (GBM), astrocytoma, and oligodendroglioma (LGG).

Obtaining sequencing data: the function in the R language package "tcgabiolinoks" is used to download from the database established by the national institute of america with the web address: https:// portal. The downloaded data included the fkpm values of RNA-seq for TCGA-GBM and TCGA-LGG, as well as relevant clinical data.

Immunohistochemistry: taking out paraffin section of tumor tissue, passing throughCarrying out gradient dewaxing and rehydration on dimethylbenzene and alcohol, and sealing endogenous peroxidase by hydrogen peroxide: 3% H₂ O₂Room temperature 10 min (dark), 5min with distilled water 2 times, antigen retrieval, PBS: after 2 times 5min, PBST containing 1% BSA was added for blocking at room temperature for 0.5 h, PBS on the slide was aspirated at one end of the section, and a suitable diluted primary antibody (DDIT4L, from Sigma) was added from the other end, covered with a humidified chamber, and incubated overnight at 4 ℃. The slides were washed 3 times with PBS (5 min/time), fresh PBS buffer was added from one end of the section, and old buffer was aspirated from the other end. Adding a secondary antibody (HRP-labeled goat anti-rabbit secondary IgG purchased from Jackson) labeled by HRP onto the section, placing the section in a humidification box, incubating for 1 hour at room temperature, washing the slide for 3 times (5 min/time) by PBS, developing DAB, observing under a mirror, stopping timely, fully washing by tap water (fine water), counterstaining by hematoxylin, washing for 30 seconds at room temperature, washing by tap water, returning blue for 15 minutes, dehydrating by gradient alcohol, enabling xylene to be transparent, and preserving the section at room temperature in a dark place for microscopic examination.

Tissue immunofluorescence: the cryosectioned tumor tissue sections were removed from the-80 ℃ freezer, rewarming at room temperature and washed once with PBS, after which the tissue section was encircled with a water-resistant pen, PBST containing 1% BSA was added to block the section at room temperature for 1 hour, PBS on the slide was aspirated off at one end of the section, and an appropriately diluted primary antibody (DDIT4L, from Sigma; GFAP, from CST; olig2, from Santa Cruz; Nestin, from STEMCELL; CD133, from Merk) was added from the other end and covered with a humidified box and incubated overnight at 4 ℃. The slides were washed 3 times with PBS (5 min/time), fresh PBS buffer was added from one end of the section, and old buffer was aspirated from the other end. Secondary antibodies labeled with different fluorophores (either Alex-488 labeled or Alex-647 labeled goat anti-rabbit/mouse secondary antibody purchased from Jackson) were added to the sections, incubated in humidified boxes for 1 hour at room temperature, and the slides were washed 3 times (5 min/time) with PBS. Place the coverslip on a paper towel and drop 1 fluorescent coverslipping piece in the center of the coverslip. The slide is placed on a cover glass, sealed with nail polish, and stored at-20 ℃ for microscopic examination.

Flow cytometry experiments: unicellular passage through 0.05% pancreatin primary cells or cell lines, counted at 5 × 10 per tube⁵The number of cells was divided, the cells were collected by centrifugation at 800g for 5 minutes, fixed with 4% PFA for 10 minutes, washed with PBS 2 times (2 minutes/time), mixed with Permeabilization Buffer (Permeabilization Buffer), incubated at 4 ℃ for 30 minutes, washed with PBS 2 times (2 minutes/time), added with primary anti-or isotype control antibodies derived from different species (DDIT4L, purchased from Sigma; Nestin, purchased from STEMCELL; CD133, purchased from Merk; IgG derived from nonimmune rabbit serum or mouse serum), incubated at 4 ℃ for 60 minutes, washed with PBS 2 times (2 minutes/time), added with secondary antibodies (goat anti-rabbit/mouse secondary antibody labeled with Alex-488 or with Alex-647, purchased from Jackson), incubated at 4 ℃ for 30 minutes, kept on ice and protected from light.

Test results and discussion

GBM is generally worse in prognosis than LGG, a low grade glioma in clinical patients. The prognosis of GBM without IDH mutation (IDH WT) is also worse than that with IDH mutation (IDH Mut). In recent years, GBM has been further functionally classified based on the expression of specific genes: proneural (Pro), neural (neural), classical (canonical), and mesenchymal (Mes). These four types have different prognosis, with the prognostics of the proto-neurogenic GBM being the best and those of the mesenchymal GBM being the worst.

To screen for molecules of potential functional significance for the prognosis or treatment of GBM, we compared RNA-seq data for gliomas with clearly different prognoses by three taxonomic approaches (i.e. GBM vs. lgg, IDH WT vs. IDH mutation and mesenchymal vs. neuroprost) using the tcga (the Cancer Genome atlas) database.

By comparison, we found a total of 53 genes whose expression was significantly upregulated (FIG. 1a), whereas DDIT4L was one of the most significant molecules upregulated in GBM (FIG. 1 b).

In the tissue staining test, it was shown that: DDIT4L expression was significantly enhanced in GBM compared to low grade gliomas (fig. 1 c). The immunofluorescence double-label and the cell flow detection show that: DDIT4L co-labeled with either glial fibrillary acidic protein (GFAP, a marker of astrocyte activation) or oligodendrocyte transcription factor-2 (Oligo2) (FIG. 1 d). Meanwhile, DDIT4L has a certain co-marker with the tumor stem cell markers CD133 and Nestin (Nestin) (FIG. 1 e).

The above results indicate that the expression level of DDIT4L has a positive correlation with the malignancy degree and prognosis of glioma, i.e., the higher the expression level of DDIT4L, the higher the malignancy degree of glioma, the worse the prognosis. Co-labeling of DDIT4L with GFAP (a marker for astrocytes), Oligo2 (a marker for oligodendrocytes), CD133 (a marker for tumor stem cells), and Nestin (a marker for tumor stem cells) in tissues, respectively, indicates that DDIT4L is localized to astrocytes or oligodendrocytes, and is localized to tumor stem cells, demonstrating its close association with gliomas.

Example 2 inhibition of glioma cell proliferation by DDIT4L

Materials and methods

Tissue specimen: tumor surgical resection specimens from Huashan Hospital, university of Compound Dane, histologically examined as Glioblastoma (GBM), astrocytoma, and oligodendroglioma (LGG).

Extraction of total protein in tissue: placing a small amount of tissue blocks in a spherical part in a homogenizer of 1-2 ml, and shearing the tissue blocks as much as possible by using clean scissors. Add 400. mu.L of protein lysate lysis solution RIPA (containing PMSF) to the homogenizer and homogenize on ice. Then slowly rotating and uniformly mixing at 4 ℃ for lysis for 2 hours, then centrifuging at 12000rpm at 4 ℃ for 5min, taking supernatant and subpackaging in 0.5ml centrifuge tubes, and placing at-20 ℃ for preservation.

Western Blot experiment: western Blot experiments were performed according to SDS-PAGE electrophoresis, membrane transfer, immunoreaction and chemiluminescence.

Tumor stem cell balling experiment: primary cells or cell lines infected with DDIT4L overexpressing lentivirus and control lentivirus, respectively, were unicellular by 0.05% pancreatin digestion, counted, and counted as 1X10⁴The cell number of (2) was inoculated in a low adhesion culture plate, cultured for 8-10 days, and 50% of fresh medium was added every 3-4 days, and photographed under an inverted phase contrast microscope every other day. Finally, counting the size and the number of the balls by ImageJ software.

Test results and discussion

Previous studies reported that hypoxia induced DDIT4L gene expression.

In patient tissues of GBM, DDIT4L is highly expressed in glioblastomas, and GBM cells are at 1% O₂The expression level of DDIT4L was significantly increased after 24 hours of culture (FIG. 2 a). By immunohistochemistry and cell flow labeling: DDIT4L was found to be co-labeled mainly with hypoxia inducible factor 1 α (HIF1 α) but not with the vascular endothelial cell marker CD31 (fig. 2b and 2 c). Overexpression of DDIT4L by infecting tumor cells in primary cells (from Washington, Inc. (11879)) with ZsGreen-PURO-LUC-labeled lentivirus (purchased from Henan biosciences (Shanghai)) recombinant for the DDIT4L gene significantly inhibited the globular growth of tumor cells (FIG. 2 d).

The above results show that: DDIT4L was highly expressed in glioblastoma and co-targeted with hypoxia inducible factor 1 α (HIF1 α), indicating that hypoxia in the tumor environment induced DDIT4L gene expression. Moreover, over-expression of DDIT4L can inhibit tumor cell proliferation and morphological changes.

Example 3 direct binding of DDIT4L to the alpha subunit of ATP synthetase

Materials and methods

The plasmid construction method comprises the following steps: the cDNA of human DDIT4L was cloned into pcDNA3.1/myc-his (+) A vector to express DDIT 4L-myc. Plasmids expressing DDIT 4L. delta.1-myc, DDIT 4L. delta.2-myc and DDIT 4L. delta.3-myc were modified from DDIT4L-myc plasmid using KOD-plus mutagenesis kit (Toyobo). The cDNA of human ATP5A was constructed in the regulated pcDNA3.1/HA-his (+) A vector because the HA tag replaced the myc tag in the pcDNA3.1/myc-his (+) A vector. Plasmids expressing ATP5A Δ 1-HA, ATP5A Δ 2-HA, ATP5A Δ 3-HA, ATP5A Δ 4-HA and ATP5A Δ 5-HA were modified from the ATP5A-HA plasmid using the KOD-plus mutagenesis kit (Toyobo). The cDNAs of human DDIT4L and human ATP5A, tagged with HA, were cloned into pGEX vectors to construct GST-DDIT4L and GST-ATP5A-HA plasmids.

Co-immunoprecipitation experiments: harvesting cells, adding an appropriate amount of cell IP lysate, performing rotary lysis at 4 ℃ for 30 minutes, centrifuging at 12000G for 30 minutes, taking the supernatant, taking a small amount of lysate for Western Blot analysis, adding 1 mu G of corresponding antibody and 10-40 mu l of protein G-beads to the remaining lysate, slowly rotating at 4 ℃ overnight, centrifuging at 4 ℃ for 5 minutes at 3000G, slowly sucking off the supernatant, washing with 400 mu l of lysate for 4-5 times, adding 1 Xloading buffer, and performing Western Blot at 100 ℃ for 10 minutes, followed by the same Western Blot step.

GST pull-down experiment: IPTG induced expression of GST-DDIT4L in Escherichia coli BL21 strain containing recombinant chloramphenicol acyltransferase protein, adding Glutathione agarose (Glutathione Sepharose) to combine with GST after obtaining lysate, washing and purifying, adding a part of the lysate to 1 Xloading buffer solution at 100 ℃ for 10 minutes for Western Blot detection; ATP5A (or various deletions) -HA fusion proteins were overexpressed in 293T cells by transfection of the corresponding plasmid, RIPA lysis; GST-DDIT4L conjugated with Sepharose is suspended in a proper amount of lysate, and 20-30 μ l ATP5A (or different deletants) -HA fusion protein lysate is added; the sample is horizontally shaken for 4-8 hours at 4 ℃, added with lysate for washing for 3 times, sucked dry, added with 1 sample buffer solution and kept at 100 ℃ for 10 minutes for Western Blot detection.

Plasmid construction experiments: the cDNA of human DDIT4L was cloned into pcDNA3.1/myc-his (+) A vector to express DDIT 4L-myc. Plasmids expressing DDIT4L1-myc, DDIT4L2-myc and DDIT4L3-myc were modified from DDIT4L-myc plasmid using KOD-plus mutagenesis kit (Toyobo). The cDNA of human ATP5A was constructed in the regulated pcDNA3.1/HA-his (+) A vector because the HA tag replaced the myc tag in the pcDNA3.1/myc-his (+) A vector. Plasmids expressing ATP5A1-HA, ATP5A2-HA, ATP5A3-HA, ATP5A4-HA and ATP5A5-HA were modified from the ATP5A-HA plasmid using the KOD-plus mutagenesis kit (Toyobo). The cDNAs of human DDIT4L and human ATP5A, tagged with HA, were cloned into pGEX vectors to construct GST-DDIT4L and GST-ATP5A-HA plasmids.

Tissue immunofluorescence experiments: the same as in example 2.

Test results and discussion

Since DDIT4L can obviously inhibit the proliferation of tumor cells, we focused the research on the action target of DDIT 4L. To investigate this, we chose the U87MG cell line (purchased from cell institute of Chinese academy) which closely approximates GBM cells.

Considering that DDIT4L has less endogenous expression in GBM and U87MG cells, we performed experiments in which myc-and His-tag-carrying DDIT4L plasmids were extracorporeally transfected by Lippo3000 reagent (supra) and then immunoprecipitated with antibodies to myc. It was found that myc antibodies could co-immunoprecipitate a 50 kDa-sized protein band (FIG. 3 a). After the protein is purified, protein mass spectrum analysis shows that the sequence of the 5-16 peptide fragment is consistent with the amino acid sequence of the alpha subunit of the human ATP synthetase, and the sequence obtained by the three experimental results can match with the amino acid sequence of 37 percent of the alpha subunit of the ATP synthetase. Co-immunoprecipitation experiments demonstrated that DDIT4L-myc can bind to the α subunit of ATP synthase (FIG. 3 a). Immunostaining also showed almost complete co-targeting of DDIT4L with ATP synthase alpha subunit in GBM tissue (fig. 3 b).

On HEK293T cells, DDIT4L was cotransformed with ATP synthase alpha subunit or beta subunit by means of Lippo3000 reagent, followed by co-immunoprecipitation to find: only the ATP synthase alpha subunit can bind to DDIT4L (fig. 3 c). Also we confirmed that the ATP synthase alpha subunit can bind to DDIT4L using GST pull-down experiments (FIG. 3 d). Further we used the purified ATP synthase alpha subunit and DDIT4L protein for direct interaction, and found that the two could be directly combined (FIG. 3 e).

To further determine the structural domain of the protein to which DDIT4L binds to the α subunit of ATP synthase, we first analyzed possible binding domains on DDIT4L by combining homologous protein structure modeling and in-silico binding modeling (the tool used was ZDCK). The analysis found that the three domains S96-C106 (SEQ ID NO:3), V125-P132(SEQ ID NO:4) and K141-R166(SEQ ID NO:5) on DDIT4L might bind to the ATP synthase alpha subunit. Three recombinant proteins respectively lacking the above domains were constructed (over-expressed on cells by means of plasmid construction; except for the above domains, they were all the same as wild-type DDIT 4L; DDIT 4L. DELTA.1, DDIT 4L. DELTA.2 and DDIT 4L. DELTA.3 in the figure lack S96-C106, V125-P132 and K141-R166 of DDIT 4L-encoding proteins, respectively), and it was confirmed by co-immunoprecipitation experiments that V125-P132 and K141-R166 (corresponding to DDIT 4L. DELTA.2 and DDIT 4L. DELTA.3, respectively) were sites where ATP synthase. alpha. subunit was bound (FIG. 3 f).

The binding region on the alpha subunit of ATP synthase (SEQ ID NO:8) was also analyzed by the same method, and 5 possible regions (SEQ ID NO: 9-13) were found: V118-V131, K132-I158, I179-G192, L471-A486 and G491-I504. The K132-I158 domain was confirmed to be the most important binding region by constructing 5 ATP synthase alpha subunits each lacking the above domains (the construction method is the same as above), and then by GST pull-down experiment (FIG. 3 g). Further using the homologous protein structure modeling and in-silicon docking model analysis, the V125-P132 domain on DDIT4L should be a functional binding site (FIG. 3 h).

From the above results, it was demonstrated that DDIT4L can directly bind to the alpha subunit of ATP synthase, and the key region for binding lies in the V125-P132 functional binding region of DDIT 4L.

Example 4 inhibition of ATP synthase Activity and mediation of apoptosis by DDIT4L

Since DDIT4L can directly bind to ATP synthase alpha subunit, we next examined the effect of DDIT4L on mitochondrial function.

Materials and methods

And (3) ATP content detection: u87 cells were plated at 5X10 per well⁴Individual cell densities were seeded in 96-well plates and transfected with DDIT4L and control vector for 30 hours in triplicate. ATP levels were measured using the ATPlite 1step routine assay (Perkinelmer, 6016739). Luminescence was read using Spectramax @ iD3(Molecular Devices) and the OD values were finally normalized by cell number.

Mitochondrial membrane potential detection: JC-10 mitochondrial membrane potential assay kit was purchased from Abcam (112134) and assayed according to the manufacturer's protocol. Cells were transfected with DDIT4L and control vectors for 30 hours, and then mitochondrial membrane potential was measured using Spectramax @ iD3(Molecular Devices).

And (3) detecting oxygen consumption: oxygen consumption was measured with a Seahorse Bioscience instrument (XF 24/XF 96, Agilent) according to the manufacturer's instruction manual, with 80-90% cell fusion. Briefly, the next day after cell seeding, cells were incubated in the absence of CO₂And then equilibrated in an incubator at 37 ℃ for 1 hour. After the compound and the peptide were added in this order under the basic conditions, the oxygen concentration in the medium was measured. Application methodThe concentrations of compound and peptide were: oligomycin A (1. mu.M, mitochondrial ATP synthase inhibitor); fccp (1 μ M, uncoupler of mitochondrial oxidative phosphorylation); derris copper (rotenone, 500nM, inhibitor of electron transport in the mitochondrial oxidative respiratory chain) and antimycin (antimycin a, 500nM, inhibitor of electron transport in the mitochondrial oxidative respiratory chain). Each condition utilizes at least three complex wells to calculate OCR.

Transmission electron microscope experiment: after 48 hours of transfection with 3. mu.g of DDIT4L overexpression or control plasmid, 5X10 were collected⁵And (4) cells. Then, the cells were first fixed in 2.5% glutaraldehyde for 2 hours. Samples were incubated with 1% OsO in the dark₄Fixation was performed for 1.5 hours. The samples were dehydrated with 30%, 50%, 70%, 80%, 95% and 100% ethanol and then twice more acetone dehydrations (100%). Finally, the samples were treated with acetone: epon812 (1: 1) for 2 hours, then Epon812 (100%) overnight. Then, ultrathin sections (70nm) were obtained, stained with uranium acetate and lead citrate, and photographed at 80kV (FEI Tecnai G2 Spirit) under TEM.

Test results and discussion

It was found that transfection with DDIT4L significantly inhibited ATP synthesis in cells and hyperpolarized mitochondrial membrane potential (FIGS. 4a and 4 b). Transfection of DDIT4L clearly inhibited both the basal oxygen consumption rate and ATP synthase-induced oxygen consumption rate of tumor cells (fig. 4 c). Transfection with DDIT4L was found to significantly enhance mitochondrial fusion using immunostaining (FIG. 4 d). Electron microscopy results also suggest that transfection with DDIT4L promotes mitochondrial fusion and intramitochondrial and outer membrane disruption and regression (fig. 4 e).

Further studies showed a significant increase in cytochrome c and activated caspase 3 in the cytoplasm of DDIT4L transfected on U87MG cells (FIG. 4 f). Transfection with DDIT4L was found to significantly promote apoptosis of GBM cells using TUNEL staining (FIG. 4 g).

On the other hand, the mTOR signaling pathway is a key signaling pathway for cell growth and autophagy. Previous studies have shown that DDIT4 and DDIT4L inhibit mTOR activity by binding to the 14-3-3 protein. Transfection of DDIT4L significantly inhibited phosphorylation of S6K, and application of mTOR agonist 3BDO partially reversed the phenomenon that transfection of DDIT4L inhibited phosphorylation of S6K (fig. 4 h).

These results indicate that the active part of DDIT4L inhibiting mTOR is involved in the process of DDIT4L inhibiting the growth of U87MG cells.

Example 5 DDIT4L and TAT-DDIT4L^V125-P132Inhibiting glioma growth

Materials and methods

Primary culture conditions of human GBM cells: primary GBM cells of patient origin were maintained in neurosphere medium (DMEM/F12, ThermoFisher, 11330032) and supplemented with B27(0.5X, ThermoFisher, 17504044), N2(0.5X, ThermoFisher, 17504048), bFGF (20ng/ml, Peprotech, 100-18B) and EGF (20ng/ml, Peprotech, 100-47), BSA (0.5 mg/ml, Sigma-Aldrich, A9418).

The PDX model construction method comprises the following steps: 48 hours after infection with ZsGreen-PURO-LUC-tagged lentivirus (MOI ═ 2; available from Hantah Biotech (Shanghai) Co., Ltd.), 2X 10 was added⁵Primary GBM cells (11879 from Huashan Hospital brain surgery patients) were implanted in the left CPU region of male Nod-SCID or Nude mice (2.0 mm injected laterally, 0mm caudal, 2.75mm ventral from pre-reg). Male litters of 8 weeks of age were used for transplantation and randomized to receive primary GBM cells expressing DDIT4L or a control vector. Survival experiments, animals were maintained until neurological symptoms appeared or 120 days post-transplantation. To monitor tumor growth, mice transplanted with primary GBM cells stably expressing firefly luciferase were monitored by bioluminescence imaging using the IVIS100 bioluminescence imaging system. To analyze the effect of peptides on GBM cell growth in vivo, a subcutaneously implanted osmotic pump was used (

1002) The method of (3), delivering a control peptide (SEQ ID NO:14) or TAT-DDIT4L V125-P132 peptide (synthesized by Gill Biochemical (Shanghai) Co., Ltd.) (dissolved in PBS) directly to the intracranial tumor site at the injection site.

Test results and discussion

Transfection of DDIT4L or intravenous administration of DDIT4L protein in a mouse model of human GBM tumor xenograft immunodeficiency in situ significantly reduced tumor size and significantly prolonged survival time in mice (fig. 5a, b, c).

As previously demonstrated in example 3, domains V125-P132 and K141-R166 on DDIT4L are sites for binding to the alpha subunit of ATP synthase (FIG. 3 f). We synthesized TAT-DDIT4L (Gill Biochemical (Shanghai) Co., Ltd.) linked to the membrane-penetrating peptide TAT (transmembrane activator protein)^V125-P132And TAT-DDIT4L^K141-R166And detecting their effect on tumor cell growth.

Experimental finding that only TAT-DDIT4L^V125-P132Showed a dose-dependent inhibition of the growth of U87MG tumor cells (fig. 5 d). TAT-DDIT4L^V125-P132The ATP synthase-induced oxygen consumption rate of U87MG tumor cells was significantly inhibited (fig. 5 e). Finally TAT-DDIT4L in a mouse model of human GBM tumor xenografted in situ immunodeficient^V125-P132It was also possible to significantly reduce the size of the tumor and significantly prolong the survival time of the mice (fig. 5f, g).

The above results demonstrate DDIT4L and TAT-DDIT4L^V125-P132Can effectively inhibit the growth of glioma and prolong the survival time of mice.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims (10)

1. Use of a DNA damage inducing transcript 4-like (DDIT4L) active for the manufacture of a product for the prevention and/or treatment of glioma and symptoms associated therewith in a subject, wherein said active is selected from the group consisting of: a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof.
2. 2. The use according to claim 1, wherein,

   the DDIT4L polypeptide has an amino acid sequence shown in SEQ ID NO. 2; and/or

   The active fragment has a fragment corresponding to a V125-P132 peptide fragment of a DDIT4L polypeptide, such as a polypeptide having an amino acid sequence shown in SEQ ID NO. 4 or 6; and/or

   The nucleic acid molecule encoding the DDIT4L polypeptide has a sequence shown as SEQ ID NO. 1; and/or

   The accelerator is selected from: a substance that increases the level of or facilitates the function of DDIT4L polypeptide or an active fragment thereof, such as an overexpression vector for DDIT4L or an active fragment thereof or a coding sequence thereof; an exogenous DDIT4L polypeptide or active fragment; naked DNA of the coding sequence of DDIT 4L; liposome-encapsulated DNA of the DDIT4L coding sequence; a precursor protein or conjugate or complex capable of being converted in vivo to DDIT4L or an active fragment thereof, for example DDIT4L or an active fragment thereof linked to a TAT peptide, such as TAT DDIT4L V125-P132 small peptide.
3. 3. The use of claim 1, wherein the glioma is a WHO grade 1 glioma, a WHO grade 2 glioma, a WHO grade 3 glioma or a WHO grade 4 glioma.
4. 4. The use according to claim 1, wherein the glioma-associated condition is selected from the group consisting of glioma-induced: headache, nausea, vomiting, epilepsy, blurred vision, vision loss, pain in the limbs, numbness, weakness in muscle strength, movement and sensation disorders, speech expression and difficulty in understanding.
5. 5. The use according to claim 1, wherein the subject is a human or non-human mammal, such as an orangutan, ape, monkey, horse, cow, sheep, dog, cat or rat.
6. 6. Use according to claim 1, wherein the product is a pharmaceutical composition or kit, for example in a form suitable for administration by a formulation selected from the group consisting of: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, intracranial administration, and intratumoral administration.
7. 7. Use according to claim 1, wherein the product further comprises other substances for the prevention and/or treatment of glioma or glioma-related symptoms, such as one or more selected from the group consisting of: temozolomide, bevacizumab avastin; alternatively, the product is used in combination with surgery, radiation therapy and/or chemotherapy.
8. 8. A product for the prevention and/or treatment of glioma and the symptoms associated therewith comprising:

   (A) a therapeutically or prophylactically effective amount of a DNA damage inducing transcript 4-like (DDIT4L) polypeptide, an active fragment thereof, a nucleic acid molecule encoding said polypeptide or fragment, or a promoter thereof, e.g., a polypeptide, an active fragment thereof, a nucleic acid molecule encoding said polypeptide or fragment, or a promoter as defined in claim 2;

   (B) a pharmaceutically acceptable carrier or excipient;

   (C) optionally, one or more other active substances for the prophylaxis and/or treatment of gliomas and their associated symptoms,

   for example, the product is characterized as defined in any one of claims 1 to 7.
9. 9. A product according to claim 8 wherein the product is a pharmaceutical composition or kit, for example in a form suitable for administration by a formulation selected from: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, DNA carried by replication-defective adenovirus or protein encoded by target gene, electroporation, nasal administration, pulmonary administration, oral administration, transdermal administration, intracranial administration, and intratumoral administration.
10. Use of a DNA damage inducing transcript 4-like (DDIT4L) active substance in the manufacture of a product for inhibiting mitochondrial metabolic activity and/or treating a disease or condition associated with mitochondrial metabolic activity, wherein the active substance is selected from the group consisting of: a DDIT4L polypeptide, an active fragment thereof, a nucleic acid molecule encoding the polypeptide or fragment, or promoters thereof.

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