Methods and pharmaceutical compositions for modulating autophagy
阅读说明：本技术 用于调节自噬的方法和药物组合物 (Methods and pharmaceutical compositions for modulating autophagy ) 是由 G·克洛伊梅 J·M·布拉沃-桑 于 2018-09-19 设计创作，主要内容包括：自噬典型地通过饥饿激活,从而允许细胞和生物体动员其能量储备。已知自噬的药理调节代表治疗潜力。本文中,发明人报道了以非常规的自噬依赖性方式从细胞释放的蛋白质,即地西泮结合抑制剂(DBI),调节自噬。特别地,发明人证实DBI抑制自噬,并且向小鼠提供重组DBI增强糖酵解,增强脂肪生成,并且抑制脂肪酸氧化。发明人表明,单克隆抗体对DBI的中和作用和引发自身抗体的免疫原性DBI衍生物的主动免疫诱导自噬,并导致代谢变化,从而增加饥饿诱导的体重减轻,减少再进食时的食物摄入,并降低响应于高热量饮食的体重增加。因此,本发明涉及基于对DBI的活性或表达的调节来调节自噬的方法和药物组合物。(Autophagy is typically activated by starvation, allowing cells and organisms to mobilize their energy stores. Pharmacological modulation of autophagy is known to represent therapeutic potential. Herein, the inventors report that a protein released from cells in an unconventional autophagy-dependent manner, i.e. a Diazepam Binding Inhibitor (DBI), modulates autophagy. In particular, the inventors demonstrated that DBI inhibits autophagy, and that providing recombinant DBI to mice enhances glycolysis, enhances lipogenesis, and inhibits fatty acid oxidation. The inventors show that the neutralizing effect of monoclonal antibodies to DBI and active immunization with immunogenic DBI derivatives eliciting autoantibodies induces autophagy and leads to metabolic changes, thereby increasing hunger-induced weight loss, reducing food intake upon refeeding, and reducing weight gain in response to a high calorie diet. Accordingly, the present invention relates to methods and pharmaceutical compositions for modulating autophagy based on modulation of the activity or expression of DBI.)
1.A method of inhibiting autophagy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that promotes DBI activity or expression.
2. The method of claim 1, wherein the subject is underweight.
3. The method of claim 2, wherein the subject has a wasting disorder.
4. The method of claim 2, wherein the subject has anorexia cachexia, senile anorexia, anorexia nervosa, cancer-related cachexia, AIDS-related cachexia, heart failure-related cachexia, cystic fibrosis-related cachexia, rheumatoid arthritis-related cachexia, kidney disease-related cachexia, Chronic Obstructive Pulmonary Disease (COPD) -related cachexia, ALS-related cachexia, renal failure-related cachexia, and other conditions associated with abnormal appetite, fat mass, energy balance, and/or involuntary weight loss.
5. The method of claim 1, wherein the subject has a disease selected from the group consisting of: cancer diseases, neurodegenerative diseases, cardiovascular diseases, infectious diseases, autoimmune diseases and/or inflammatory diseases.
6. The method of claim 1, wherein the agent that promotes the activity of DBI consists in a polypeptide comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO. 1, or ii) an amino acid sequence having at least 80% identity to the amino acid sequence of amino acid residue 17 to amino acid residue 50 of SEQ ID NO. 1, or iii) an amino acid sequence having at least 80% identity to the amino acid sequence of amino acid residue 33 to amino acid residue 50 of SEQ ID NO. 1, or iv) an amino acid sequence having at least 80% identity to the amino acid sequence of amino acid residue 43 to amino acid residue 50 of SEQ ID NO. 1.
7. The method of claim 1, wherein the agent that promotes expression of DBI is a nucleic acid molecule encoding the polypeptide of claim 6.
8. The method of claim 7, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least 50% identity to SEQ ID NO 2.
9. The method of claim 1, wherein the agent that promotes the activity of DBI is a small organic molecule or peptidomimetic that mimics the activity of DBI.
10. A method of stimulating autophagy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that inhibits the activity or expression of DBI.
11. The method of claim 10, wherein the subject is overweight.
12. The method of claim 11, wherein the subject is suffering from obesity.
13. The method of claim 10, wherein the subject has type 2 diabetes or metabolic syndrome.
14. The method of claim 10, wherein the subject has cancer; neurodegenerative diseases; infectious diseases; pulmonary diseases, such as emphysema, cystic fibrosis; liver disease, pancreatitis or proteinopathy.
15. The method of claim 14, comprising administering to the subject having cancer a therapeutically effective amount of an agent that inhibits the activity or expression of DBI and a therapeutically effective amount of a chemotherapeutic agent, wherein the agent that inhibits the activity or expression of DBI is administered prior to the chemotherapeutic agent.
16. The method of claim 10, wherein the agent that inhibits the activity of DBI is an antibody or aptamer against DBI.
17. The method of claim 16, wherein the antibody is directed against a fragment consisting of the amino acid sequence of amino acid residue 43 to amino acid residue 50.
18. The method of claim 16, wherein the antibody is a monoclonal chimeric antibody, a monoclonal humanized antibody, or a monoclonal human antibody.
19. The method according to claim 10, wherein the agent inhibiting the expression of DBI is an expression inhibitor, such as siRNA, endonuclease, antisense oligonucleotide or ribozyme.
20. The method of claim 10, wherein the agent that inhibits the activity of DBI consists in a vaccine composition suitable for eliciting neutralizing autoantibodies against DBI when administered to a subject.
21. The method of claim 10, wherein the vaccine composition comprises an antigen as a polypeptide comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO. 1, or ii) an amino acid sequence having at least 80% identity to the amino acid sequence of amino acid residue 17 to amino acid residue 50 of SEQ ID NO. 1, or iii) an amino acid sequence having at least 80% identity to the amino acid sequence of amino acid residue 33 to amino acid residue 50 of SEQ ID NO. 1, or iv) an amino acid sequence having at least 80% identity to the amino acid sequence of amino acid residue 43 to amino acid residue 50 of SEQ ID NO. 1.
22. The method of claim 10, wherein the polypeptide is conjugated to a carrier protein that is generally foreign enough to elicit a strong immune response to the vaccine.
23. The method of claim 10, wherein the vaccine composition comprises an adjuvant.
24. A method of screening for a compound suitable for modulating autophagy comprising: i) providing a candidate compound, ii) determining whether said candidate compound is capable of modulating the activity or expression of DBI, and iii) positively selecting said candidate compound capable of modulating the activity or expression of DBI.
25. A method of determining whether a subject is at risk for weight regulation, comprising: i) determining the level of DBI in a blood sample obtained from the subject, ii) comparing the level determined in step i) with a predetermined reference value, and iii) concluding that the subject is at risk of weight regulation when a difference between the level determined in step i) and the predetermined reference value is determined.
26. A method of treating nonalcoholic fatty liver disease (NAFLD) in a subject thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits the activity or expression of DBI.
27. The method of claim 16, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).
Technical Field
The present invention relates to methods and pharmaceutical compositions for modulating autophagy.
Background
Autophagy ("autophagy") constitutes one of the most dramatic, although finely regulated, phenomena in cell biology and plays a key role in maintaining cellular and biological homeostasis by promoting the renewal of cytoplasmic structures and allowing cells to adapt to changing and stressful conditions, including nutritional deficiencies (1, 2). Several cell secretions free of leader proteins, which can only be released by non-canonical pathways bypassing the golgi, are closely associated with autophagy (3-7). One such protein is the phylogenetic ancient factor known as diazepam binding protein (DBI) or acyl-coa (coa) binding protein (ACBP) (3, 4). The DBI of human or mouse is 87 amino acids (10kDa)Small proteins with two completely different functions, namely as intracellular ACBP (where it binds to a long-chain acyl-CoA molecule) and as extracellular DBI (where the intact protein or its cleavage product, triacontateneuronectide [ TTN, residues 17-50)]And octadecaneuropeptide [ ODN, residues 33-50]Can be used for treating gamma-aminobutyric acid (GABA) type A receptorAR) interacts with and modulates its activity as a GTP protein-coupled receptor (GPCR) (8-10). DBI and its proteolytic fragments also bind to Peripheral Benzodiazepine Receptors (PBR) (11-13) as well as unidentified GPCRs (ODN-GPCRs) (14-17). However, the role of DBI secretion in feedback regulation of autophagy has never been investigated.
Brief description of the invention
The present invention relates to methods and pharmaceutical compositions for modulating autophagy. In particular, the invention is defined by the claims.
Detailed Description
Autophagy is typically activated by starvation, allowing cells and organisms to mobilize their energy stores. Here, the inventors report that proteins released from cells in an unconventional autophagy-dependent manner, namely Diazepam Binding Inhibitor (DBI), also known as acyl-coa binding protein (ACBP), modulate autophagy at three levels. First, autophagy causes secretion of DBI, thereby depleting this autophagy factor from the cell (autocrine regulation). Second, autophagy results in the accumulation of DBI in the extracellular space, allowing DBI to act on other cells to inhibit autophagy (paracrine regulation). Third, circulating DBI stimulates eating behavior, thereby eliminating the major cause of autophagy induction (endocrine regulation). In humans, obese persons have elevated plasma DBI levels. Additional provision of recombinant DBI to mice enhanced glycolysis, enhanced lipogenesis and inhibited fatty acid oxidation. The inventors have also devised three strategies for neutralizing DBI, namely active immunization by inducing systemic knockdown, passive immunization and priming of autoantibodies by immunogenic DBI derivatives. These strategies are beneficial to increase weight loss due to hunger and to reduce metabolic changes in food intake after refeeding.
General definition:
as used herein, the terms "subject", "individual" or "patient" are used interchangeably and refer to any subject, particularly a human, in need of diagnosis, treatment or therapy. Other subjects may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments, the subject is a human.
Unless otherwise indicated, the term "autophagy" refers to macroscopic autophagy, which is a metabolic process involving the degradation of components of the cell itself (such as long-lived proteins, protein aggregates, organelles, cell membranes, organelle membranes, and other cellular components). Mechanisms of autophagy may include: (i) forming a membrane around the target region of the cell, separating the contents from the remainder of the cytoplasm, (ii) fusion of the resulting vesicle with lysosomes, followed by degradation of the vesicle contents. The term autophagy can also refer to one of the mechanisms by which starved cells redistribute nutrients from unnecessary processes to more important processes. Also, for example, autophagy can inhibit the development of certain diseases and act as a protective against intracellular pathogen infection. Acute, intermittent, or continuous stimulation of autophagy can delay aging and aging-related diseases, including arteriosclerosis, cardiac insufficiency, cancer, and neurodegenerative diseases. Stimulation of autophagy can also reduce high fat or high sugar diet or high salt induced weight gain, obesity, metabolic syndrome, hypertension and diabetes.
As used herein, the term "body mass index" has its ordinary meaning in the art and refers to the ratio calculated as weight per height in square meters (kg/m)2). BMI provides a simple method to assess how much an individual's weight differs from the normal or expected weight of a person of his or her height. A common definition of the BMI class is as follows: starvation-BMI less than 15kg/m2(ii) a Body weight deficiency-BMI less than 18.5kg/m2(ii) a Ideally the BMI is 18.5-25kg/m2(ii) a overweight-BMI 25-30kg/m2(ii) a The obesity-BMI is 30-40kg/m2(ii) a Morbid obesity-BMI greater than 40kg/m2. Although simple, BMI methods that characterize a person's body weight are not always accurate. For example, BMI does not consider the following factors: such as skeleton size, muscle or, for example, weight of bone, cartilage and water between individualsThe ratio is varied. Therefore, the accuracy of BMI associated with actual levels of body fat mass may be distorted by factors such as fitness level, muscle mass, bone structure, gender, and race. Also, short-stature and elderly tend to have lower BMI values. However, it is believed that one skilled in the art (e.g., a physician) will be able to consider these factors in assessing BMI for any given individual.
As used herein, the term "cancer" has its ordinary meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and blood vessels. The term "cancer" also encompasses both primary and metastatic cancers. Examples of cancers that may be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells from: bladder, blood, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. Furthermore, the cancer may be specifically of the following histological types, but is not limited to these: tumor, malignant; cancer; cancer, undifferentiated; giant cell and spindle cell cancers; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinomas, malignant; bile duct cancer; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial colonic polyps; a solid cancer; carcinoid tumor, malignant; bronchoalveolar carcinoma; papillary adenocarcinoma; a cancer of the chromophobe; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic granulosa cancer; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-enveloped, hard-set cancers; adrenocortical carcinoma; endometrioid carcinoma; skin adjunct cancer; hyperhidrosis carcinoma; sebaceous gland cancer; earwax; adenocarcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease, breast cancer; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; sarcoma, malignant; granulocytoma, malignant; and blastoma (robustoma), malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipocytoma, malignant; paraganglioma, malignant; external paraganglioma of mammary gland, malignant; pheochromocytoma; hemangiospherical sarcoma; malignant melanoma; achrominomatous melanoma; superficial diffusible melanoma; malignant melanoma in giant pigmented nevi; epithelial-like cell melanoma; blue nevus, malignant; a sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; mixed tumor, malignant; a Mullerian mixed tumor; nephroblastoma; hepatoblastoma; a carcinosarcoma; phyllomas, malignant; burlen tumor (brenner tumor), malignant; phylloid tumors, malignant; synovial sarcoma; mesothelioma, malignant; clonal cell tumors; an embryonic carcinoma; teratoma, malignancy; ovarian goiter, malignant; choriocarcinoma; middle kidney tumor, malignant; angiosarcoma; vascular endothelioma, malignant; kaposi's sarcoma; vascular endothelial cell tumor, malignant; lymphangioleiomyosarcoma; osteosarcoma; (ii) a cortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumors, malignant; amelogenic cell dental sarcoma; ameloblastoma, malignant; amelogenic cell fibrosarcoma; pineal, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; a plasma astrocytoma; fibroid astrocytoma; astrocytomas; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectoderm; cerebellar sarcoma; ganglion cell blastoma; neuroblastoma; retinoblastoma; olfactive neurogenic tumors; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granulocytoma, malignant; malignant lymphoma; hodgkin's disease; hodgkin lymphoma; granuloma paratuberis; malignant lymphoma, small lymphocytes; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other designated non-hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic granulocytic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
The terms "polypeptide" and "protein" are used interchangeably and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences; a fusion protein having heterologous and homologous signal sequences; a fusion protein with or without an N-terminal methionine residue; immunolabeling proteins, and the like.
As used herein, the term "DBI" has its ordinary meaning in the art and refers to a diazepam binding inhibitor, an acyl-CoA binding protein encoded by the DBI gene (gene ID: 1622). The term is also referred to as EP; ACBP; ACBD 1; and CCK-RP. An exemplary human amino acid sequence is represented by the NCNI reference sequence NP-001073331.1 (SEQ ID NO:1) (acyl-CoA binding protein isoform 1). An exemplary human nucleic acid sequence is represented by the NCNI reference sequence NM-001079862.2 (SEQ ID NO:2) (acyl-CoA binding protein isoform 1).
SEQ ID NO:1
MSQAEFEKAA EEVRHLKTKP SDEEMLFIYG HYKQATVGDI NTERPGMLDF TGKAKWDAWNELKGTSKEDA MKAYINKVEE LKKKYGI
SEQ ID NO:2
As used herein, the term "DBI activity" refers to any biological activity of DIB, including: inhibiting autophagy, inducing hypoglycemia, stimulating food intake, stimulating weight gain, reducing fatty acid oxidation, up-regulating glucose transporters, up-regulating PPARG, stimulating glucose intake, stimulating glycolysis, or stimulating lipogenesis.
As used herein, the term "treatment" refers to both prophylactic or preventative treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting or suspected to have contracted a disease as well as patients who are ill or have been diagnosed with a disease or medical condition, and includes inhibition of clinical relapse. A treatment can be administered to a subject having a medical condition or who may ultimately have a condition, in order to prevent, cure, delay onset of, reduce severity of, or ameliorate one or more symptoms of the condition or recurring condition, or to extend survival of the subject beyond that which would be expected in the absence of such treatment. "treatment regimen" refers to the mode of treatment of a disease, e.g., the dosage mode used during treatment. The treatment regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of a disease. The overall goal of the induction regimen is to provide high levels of drug to the patient during the initial phase of the treatment regimen. The induction regimen may employ a (partial or complete) "loading regimen" which may include administering a drug at a greater dose than the physician employs during the maintenance regimen, administering the drug more frequently than the physician administers during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during treatment of a disease, e.g., to maintain the patient in remission for an extended period of time (months or years). Maintenance regimens may employ continuous therapy (e.g., administration of drugs at regular intervals (e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinuation of therapy, intermittent therapy, therapy at relapse, or therapy at achievement of certain predetermined criteria [ e.g., clinical manifestation of disease, etc.)).
"therapeutically effective amount" means a sufficient amount of an agent of the invention to achieve a therapeutic effect. It will be understood, however, that the total daily amount of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the condition being treated and the severity of the condition; the activity of the particular compound employed; the particular composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of secretion of the particular compound used; the duration of the treatment; drugs used in combination or coincidental with the particular compound employed; and similar factors well known in the medical arts. For example, it is within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product may vary within a wide range of 0.01-4,000mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500 and 1000mg of the active ingredient for the symptomatic adjustment of the dose to the subject to be treated. Typically, the medicament contains from about 0.01mg to about 1000mg of the active ingredient. The effective amount of the drug is typically provided at a dosage level of from 0.0002mg per kg body weight to about 50mg per kg body weight per day, especially from about 0.001mg per kg to 10mg per kg body weight per day.
Methods of inhibiting autophagy:
accordingly, a first object of the present invention relates to a method of inhibiting autophagy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an agent that promotes the activity or expression of DBI.
In some embodiments, the method of inhibiting autophagy according to the invention is particularly suitable for stimulating appetite and thus increasing body weight. In particular, the method of the invention is also particularly suitable for promoting glucose uptake and adipogenesis. Thus, the methods of the invention are particularly useful for treating various diseases as described below.
Thus, in some embodiments, the subject is underweight. As used herein, the term "insufficient body weight" refers to a subject with a body mass index of less than 18.5.
The lack of body weight may be due to a variety of causes, such as rapid metabolism, poor/inadequate diet or hunger (malnutrition), malabsorption due to defective gut function, endocrine disorders (e.g. type I diabetes), psychological problems (such as anorexia nervosa, somatoform disorders, stress and anxiety) and weight loss due to chronic diseases and aging. In general, the root cause of the underweight must be treated, and underweight may also be a health hazard, and must therefore be treated as such. In fact, people with insufficient body weight are often physically weak, have a weak immune system, and are at higher risk of diseases such as osteoporosis, heart disease, and vascular disease. In addition, in women, insufficient body weight can lead to delayed sexual development, delayed amenorrhea or complications during pregnancy.
In some embodiments, the subject has a wasting disorder (wasting disorder). As used herein, the term "wasting disorder" has its general meaning in the art and includes, but is not limited to, anorexia cachexia, anorexia nervosa, cancer-related cachexia, AIDS-related cachexia, heart failure-related cachexia, cystic fibrosis-related cachexia, rheumatoid arthritis-related cachexia, kidney disease-related cachexia, Chronic Obstructive Pulmonary Disease (COPD) -related cachexia, ALS-related cachexia, renal failure-related cachexia, and other disorders associated with abnormal appetite, fat mass, energy balance, and/or involuntary weight loss.
In some embodiments, the subject has "cachexia". As used herein, the term "cachexia" is used in the condition of wasting body mass due to loss of body fat and muscle mass. Generally, cachexia can be associated with and caused by the following conditions: such as cancer, acquired immunodeficiency syndrome (AIDS), heart disease, infectious disease, shock, burn, endotoxemia, organ inflammation, surgery, diabetes, collagen disease, radiation therapy and chemotherapy. In many of these diseases, cachexia may contribute significantly to morbidity or mortality. Another specific group of individuals susceptible to developing cachexia are those who have undergone gastrectomy, such as may be performed on gastric cancer and ulcer patients.
In some embodiments, the subject has anorexia. As used herein, the term "anorexia" has its ordinary meaning in the art and refers to any eating disorder characterized by a significant reduction in appetite or complete aversion to food. In some embodiments, the subject has anorexia nervosa. Typically, the BMI of a subject suffering from anorexia nervosa is less than 17.5kg/m2。
Accordingly, the present invention relates to a method of treating a patient exhibiting one or more wasting disorders such as anorexia cachexia, anorexia nervosa, cancer-related cachexia, AIDS-related cachexia, heart failure-related cachexia, cystic fibrosis-related cachexia, rheumatoid arthritis-related cachexia, kidney disease-related cachexia, COPD-related cachexia, ALS-related cachexia, renal failure-related cachexia or hip fracture, as well as reducing mortality and morbidity in critically ill patients, comprising administering to said patient in need of such treatment a therapeutically effective agent that promotes DBI activity or expression.
In some embodiments, the subject has a disease selected from the group consisting of: cancer diseases, neurodegenerative diseases, cardiovascular diseases, infectious diseases, autoimmune diseases and/or inflammatory diseases.
In some embodiments, the subject has cancer. In particular, autophagy appears to be essential for tumor progression, providing the tumor with the structure and energy required for increased metabolism. Modulating the metabolic environment of a tumor by administering an agent that promotes DBI activity or expression, alone or in combination with a chemotherapeutic agent, inhibits basal and starvation-induced autophagy, thereby sensitizing tumor cells to death. Thus, agents that promote the activity or expression of DBI of the invention would be useful in the treatment of advanced cancers. In some embodiments, the cancer is an autophagy competent cancer (autophagy competence cancer). As used herein, the term "autophagy-competent cancer" refers to a cancer in which autophagy is likely to occur. In some embodiments, no ATG5 or ATG7 defects are detected. In the context of the present invention, the term "ATG 5 or ATG7 deficient" means that the tumor cells of the subject or a part thereof have ATG5 or ATG7 dysfunction, low expression or no expression of the ATG5 or ATG7 gene. Typically, the defect is attributable to a mutation in the ATG5 or ATG7 genes, such that the pre-ARNm is degraded by the NMD (nonsense-mediated decay) system. Typically, the defect may also be caused by a mutation, such that the protein is misfolded and degraded by the proteasome. The defect may also be caused by the loss of a functional mutation that causes protein dysfunction. The defect may also result from epigenetic control of gene expression (e.g., methylation), such that the gene is less expressed in the cells of the subject. The defect may also be due to inhibition of the ATG5 or ATG7 gene induced by a specific signaling pathway. The defect may also be caused by a mutation in the nucleotide sequence controlling the expression of ATG5 or ATG7 gene.
In some embodiments, the subject has a neurodegenerative disease for which inhibition of autophagy would be appropriate. Typically, the subject has amyotrophic lateral sclerosis. As used herein, the term "Amyotrophic Lateral Sclerosis (ALS)" includes the known series of neurodegenerative syndromes named as classical (Charcot's) ALS, Lou Gehrig's disease, Motor Neuron Disease (MND), Progressive Bulbar Palsy (PBP), Progressive Muscular Atrophy (PMA), Primary Lateral Sclerosis (PLS), bulbar paroxysmal ALS, spine paroxysmal ALS, and ALS involving multiple systems (wijeseekera LC and leighpn.
In some embodiments, the subject has sarcopenia. As used herein, the term "sarcopenia" refers to a gradual reduction in skeletal muscle mass caused by aging, which may directly result in a reduction in muscle strength, resulting in a reduction and impairment of various bodily functions.
Promoting DBI activity or tableThe reagent of Dada:
in some embodiments, the agent that promotes DBI activity is a polypeptide having at least 80% identity to the sequence of SEQ ID No. 1 or a fragment thereof.
According to the invention, a first amino acid sequence having at least 80% identity to a second amino acid sequence means that the first sequence has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the second amino acid sequence. Sequence identity is often measured in terms of percent identity (or similarity or homology). The higher the percentage, the more similar the two sequences. Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in the following: smith and Waterman, adv.appl.math, 2:482,1981; needleman and Wunsch, J.mol.biol.,48:443,1970; pearson and Lipman, proc.natl.acad.sci.u.s.a.,85:2444,1988; higgins and Sharp, Gene,73: 237-; higgins and Sharp, CABIOS,5: 151-; copet et al Nuc. acids Res.,16:10881-10890, 1988; huang et al, Comp.appls biosci, 8: 155-; and Pearson et al, meth.mol.biol.,24: 307-. For example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17,1989) or LFASTA (Pearson and Lipman,1988) can be used to perform sequence comparisons (Internet)
As used herein, the term "fragment" refers to a physically contiguous portion of the primary structure of a polypeptide (i.e., SEQ ID NO: 1). In some embodiments, a fragment comprises at least 8 contiguous amino acids of
In some embodiments, a fragment consists of an amino acid sequence from amino acid residue 17 to amino acid residue 50 (i.e., triacontateneuropeptide or TTN).
In some embodiments, a fragment consists of an amino acid sequence from amino acid residue 33 to amino acid residue 50 (i.e., an octadecapeptide or ODN).
In some embodiments, a fragment consists of an amino acid sequence from amino acid residue 43 to amino acid residue 50 (i.e., an octapeptide or OP).
Thus, in some embodiments, the agent that promotes DBI activity consists in a polypeptide comprising:
-an amino acid sequence having at least 80% identity with SEQ ID No. 1, or
-an amino acid sequence having at least 80% identity with the amino acid sequence from amino acid residue 17 to
-an amino acid sequence having at least 80% identity with the amino acid sequence from amino acid residue 33 to
-an amino acid sequence having at least 80% identity with the amino acid sequence from amino acid residue 43 to
In some embodiments, the polypeptide of the invention is fused to at least one heterologous polypeptide to form a fusion protein. In some embodiments, the polypeptide of the invention is fused to the N-terminus of the heterologous polypeptide, either directly or through a spacer at its C-terminus, or a spacer at its N-terminus is fused to the C-terminus of the heterologous polypeptide. The term "directly" as used herein refers to the fusion of the terminal (N-or C-terminal) (first or last) amino acid of a polypeptide of the invention with the terminal (N-or C-terminal) (first or last) amino acid of a heterologous polypeptide. In other words, in this embodiment, the last amino acid at the C-terminus of the polypeptide is directly linked to the first amino acid at the N-terminus of the heterologous polypeptide by a covalent bond, or the first amino acid at the N-terminus of the polypeptide is directly linked to the last amino acid at the C-terminus of the heterologous polypeptide by a covalent bond. The term "spacer" as used herein refers to a sequence of at least one amino acid linking a polypeptide of the invention to a heterologous polypeptide. Such spacers may be used to prevent steric hindrance. Typically, the spacer comprises 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19 or 20 amino acids.
In some embodiments, the polypeptides of the invention are fused to a signal sequence. The signal sequence may be used to facilitate secretion and isolation of secreted or other proteins of interest. Typically, the signal sequence is characterized by a core of hydrophobic amino acids that are normally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature protein as they pass through the secretory pathway.
In some embodiments, the fusion protein according to the invention is an immunoadhesin. As used herein, the term "immunoadhesin" refers to antibody-like molecules that bind the binding specificity of the polypeptides of the invention to the effector function of an immunoglobulin constant domain. Structurally, the immunoadhesins comprise a fusion of a polypeptide of the invention and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM. The immunoglobulin sequence is typically, but not necessarily, an immunoglobulin constant domain (Fc region). Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from human protein sequences of desired specificity linked to appropriate human immunoglobulin hinge and constant domain (Fc) sequences, the entire human component can be used to achieve the target binding specificity. Such immunoadhesins are minimally immunogenic to the patient and are safe for long-term or repeated use. In some embodiments, the Fc region is a native sequence Fc region. In some embodiments, the Fc region is a variant Fc region. In another embodiment, the Fc region is a functional Fc region. As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The adhesion portion of the immunoadhesin and the immunoglobulin sequence portion may be joined by a minimal linker. The immunoglobulin sequence is typically, but not necessarily, an immunoglobulin constant domain. The immunoglobulin moiety in the chimeras of the invention may be obtained from IgG1, IgG2, IgG3 or IgG4 subtype, IgA, IgE, IgD or IgM, but is typically IgG1 or
The polypeptides of the invention are produced by any technique known per se in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination. For example, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce the polypeptide by standard techniques for producing amino acid sequences. For example, they can be synthesized using well-known solid phase methods, preferably using commercially available peptide synthesis equipment (such as that manufactured by Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions. Alternatively, the polypeptides of the invention may be synthesized by recombinant DNA techniques as are currently well known in the art. For example, these fragments may be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly) peptide into an expression vector and introducing such vector into a suitable eukaryotic or prokaryotic host in which the desired polypeptide will be expressed, from which they are then isolated using well-known techniques.
In some embodiments, it is contemplated that the polypeptides of the invention used in the treatment methods of the invention may be modified to improve their therapeutic efficacy. Such modifications of therapeutic compounds can be used to reduce toxicity, increase circulation time, or alter biodistribution. For example, toxicity of potentially important therapeutic compounds can be significantly reduced by combination with a variety of drug carrier media that alter biodistribution. A strategy to improve the pharmaceutical feasibility is to utilize water soluble polymers. Various water-soluble polymers have been shown to alter biodistribution, improve cellular uptake patterns, alter permeability through physiological barriers; and change the rate of removal from the body. To achieve targeted or sustained release effects, water-soluble polymers have been synthesized that contain a drug moiety as an end group, as part of the backbone, or as a pendant group on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier in view of its high biocompatibility and easy modification. Attachment to various drugs, proteins and liposomes has been shown to improve residence time and reduce toxicity. PEG can be coupled to the active agent through hydroxyl groups at the chain ends and other chemical methods; however, PEG itself is limited to a maximum of two active agents per molecule. In another approach, copolymers of PEG and amino acids were explored as novel biomaterials that would retain the biocompatibility of PEG, but have the added advantage of numerous attachment points per molecule (providing greater drug loading) and can be synthetically designed to suit a variety of applications.
In some embodiments, the agent that promotes DBI expression is a nucleic acid molecule encoding a polypeptide as described above. As used herein, the term "nucleic acid molecule" has its ordinary meaning in the art and refers to a DNA or RNA molecule. However, the term captures the sequence of any known base analog including DNA and RNA, such as but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, azidocytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyl uracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseuduroidine, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyluracil, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutyloxypyrimidine, pseudouracil, quinine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, uracil, Quinine, 2-thiocytosine and 2, 6-diaminopurine.
In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 50% identity to SEQ ID No. 2. According to the present invention, a first nucleic acid sequence having at least 50% identity to a second nucleic acid sequence means that the first sequence has 50 to the second nucleic acid sequence; 51; 52; 53; 54, a first electrode; 55; 56; 57; 58; 59; 60, adding a solvent to the mixture; 61; 62, a first step of mixing; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78, a nitrogen source; 79; 80; 81; 82; 83; 84; 85 parts by weight; 86; 87; 88; 89; 90, respectively; 91; 92; 93; 94; 95; 96; 97, a stabilizer; 98, respectively; 99 or 100% identity.
In some embodiments, the nucleic acid molecules of the invention are contained in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. Typically, the vector is a viral vector, which is an adeno-associated virus (AAV), a retrovirus, a bovine papilloma virus, an adenoviral vector, a lentiviral vector, a vaccinia virus, a polyoma virus or an infectious virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. The AAV vector may have one or more AAV wild-type genes, preferably rep and/or cap genes, deleted in whole or in part, but retain functional ITR flanking sequences. Retroviruses may be selected as gene delivery vehicles because of their ability to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a broad spectrum of species and cell types, and package in specific cell lines. To construct retroviral vectors, nucleic acids encoding a gene of interest are inserted into the viral genome in place of certain viral sequences to produce replication-defective viruses. For the production of virions, packaging cell lines were constructed which contained gag, pol and/or env genes but no LTRs and/or packaging components. When the cDNA-containing recombinant plasmid is introduced into the cell line (e.g., by calcium phosphate precipitation) along with the retroviral LTR and the packaging sequence, the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted into the culture medium. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. Lentiviruses are complex retroviruses which contain, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural functions. The higher complexity enables the virus to regulate its life cycle, such as during a potential infection. Some examples of lentiviruses include human immunodeficiency virus (
In some embodiments, the agent that promotes the activity of DBI is a small organic molecule or peptidomimetic that mimics the activity of DBI. As used herein, the term "small organic molecule" refers to a molecule of comparable size to organic molecules typically used in pharmaceuticals. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000Da, more preferably up to 2000Da, and most preferably up to about 1000 Da. As used herein, the term "peptidomimetic" refers to any molecule whose elemental element (pharmacophore) mimics a native peptide or protein in 3D space and which retains the ability to interact with a biological target and produce the same biological effect. Peptoids include small protein-like chains designed to mimic peptides, which are typically obtained by modification of existing peptides or by designing analogous systems for mimetic peptides, such as peptoids and β -peptides. Regardless of the method employed, the altered chemical structure is designed to beneficially modulate a molecular property, e.g., an increase or decrease in stability or biological activity. Modifications according to alterations involving peptides (which would not occur naturally) include, but are not limited to, backbone alterations and incorporation of unnatural amino acids. As used herein, the term "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.
Methods of stimulating autophagy:
accordingly, a first object of the present invention relates to a method of stimulating autophagy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an agent that inhibits the activity or expression of DBI.
In some embodiments, the method of stimulating autophagy according to the invention is particularly suitable for suppressing appetite and thus weight loss. The method is also particularly useful for reducing blood glucose and adipogenesis. Thus, the methods of the invention are particularly useful for treating various diseases as described below.
In some embodiments, the subject is overweight. In particular, the subject is obese. Obese refers to a healthy subject having a BMI greater than or equal to 30kg/m2Or the BMI of a subject with at least one complication is greater than or equal to 27kg/m2The case (1). An "obese subject" is a subject with a BMI greater than or equal to 30kg/m2Or a BMI of greater than or equal to 27kg/m2A subject with at least one complication. A "subject at risk of obesity" is a BMI of 25kg/m2To less than 30kg/m2A healthy subject of (2), or a BMI of 25kg/m2To less than 27kg/m2A subject with at least one complication. The increased risk associated with obesity may occur in asian progeny where BMI is low. In Asia and Asia-Pacific countries (including Japan), "obesity" means that the subject's BMI is greater than or equal to 25kg/m2The case (1). In these countries, "obese subjects" means BMI greater than or equal to 25kg/m2Subject suffering from at least one obesity-induced or obesity-related complication in need of or ameliorated by weight loss. In these countries, "subjects at risk of obesity" means BMI greater than 23kg/m2To less than 25kg/m2The person of (1).
In some embodiments, the subject has
In some embodiments, the subject has metabolic syndrome. As used herein, the term "metabolic syndrome" refers to a subject characterized by three or more symptoms of: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and high fasting glucose. The criteria for these symptoms are defined in the third report by the national cholesterol education program panel on the detection, assessment and treatment of adult high blood cholesterol (Ford, E s.et al 2002).
In some embodiments, the subject has cancer. Although the underlying mechanism has not been elucidated, starvation prior to chemotherapy (the most effective autophagy-inducing physiological stimulus capable of inducing autophagy systemically) has been shown to significantly improve therapeutic efficacy and limit tumor growth. Furthermore, tumors with over-activation of PI3K have been demonstrated to be resistant to dietary restriction, suggesting an important role for autophagy in the process of chemosensitization. The present invention may result in less aggressive and equivalent treatments based on the timely administration of the agents of the invention. Thus, another object of the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent of the present invention and a therapeutically effective amount of a chemotherapeutic agent, wherein the agent of the present invention is administered prior to the chemotherapeutic agent. In some embodiments, prior to administration of the
In some embodiments, the subject has a neurodegenerative disease. Examples of neurodegenerative diseases include, but are not limited to, Adrenomyelodystrophy (ALD), Alexander's disease, Alper's disease, Alzheimer's disease, muscular dystrophyAmyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia telangiectasia, Batten's disease (also known as Spielmeyer-Vogt-
In some embodiments, the subject has an infectious disease. The autophagy process is actively involved in a multi-vascular defense against microorganisms, helping to eliminate them by selectively delivering them to degraded lysosomes (a process known as xenophagocytosis) or by delivering microbial nucleic acids to the lysosomal compartment (followed by activation of innate and adaptive immunity). Clinically relevant pathogens are degraded in vitro by xenophagocytosis; wherein bacteria are present, such as Streptococcus pyogenes group A, Mycobacterium tuberculosis, Shigella flexneri, Salmonella enterica, Listeria monocytogenes; viruses, such as herpes simplex virus type 1 (HSV 1) and parasites, such as toxoplasma gondii. In addition, in vivo evidence suggests that autophagy genes have protective effects against a variety of pathogens, including Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica, Toxoplasma gondii,
In some embodiments, the subject has emphysema. Mutations in protein alpha 1-antitrypsin cause emphysema, a disease characterized by accumulation of aggregated forms of the mutant protein. For other proteinopathies, inducing autophagy by administering an agent of the invention (e.g., HC, UK-5099) can reduce symptoms.
In some embodiments, the subject has cystic fibrosis. Recent preclinical studies found that impaired aggregate clearance of mutant CTFR, due to dysfunctional aggregate autophagy, led to the pathogenicity of cystic fibrosis. Induction of autophagy mediated by administration of an agent of the invention may represent a suitable strategy.
In some embodiments, the subject has liver disease. The potential effect of autophagy in vivo was discovered from liver studies, highlighting the important role of autophagy in liver physiology.
Accordingly, another object of the present invention relates to a method of treating non-alcoholic fatty liver disease in a subject thereof, comprising administering to said subject a therapeutically effective amount of an agent that inhibits the activity or expression of DBI.
As used herein, the term "non-alcoholic fatty liver disease" or "NAFLD" has its general meaning in the art and refers to a cause of fatty liver that occurs when fat is deposited in the liver rather than due to excessive alcohol consumption. Non-alcoholic fatty liver disease (NAFLD) represents one of the most common and serious pathologies, especially in obese and diabetic patients, but there is far from no specific therapy available. NAFLD is defined as the accumulation of fat in the liver, but is not a secondary consequence of alcohol intake. The autophagy promoting ability of the agents of the invention can be used for the treatment of NAFLD of different etiology: selective degradation of TG droplets (lipophagocytosis), inhibition of the lipogenic pathway (i.e. BTC inhibits citrate export from mitochondria). In contrast, the autophagy inducer perhexiline plays an adaptive and protective role in ALD, conferring hepatocyte protection and inhibiting adipocyte differentiation after alcoholism.
NAFLD can be sub-classified as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver (NAFL). Non-alcoholic fatty liver disease (NAFL) is one of NAFLD, and is a condition in which fat accumulates in hepatocytes. The risk of NAFL developing cirrhosis is minimal. Nonalcoholic steatohepatitis (NASH), the more extreme form of NAFLD, is thought to be the leading cause of liver fibrosis and cirrhosis of unknown etiology. The main features of NASH are fat in the liver as well as inflammation and damage. NASH can be severe and can lead to fibrosis and cirrhosis, where the liver is permanently damaged and scarred and can no longer function properly. Most patients with NAFLD have little or no symptoms. The patient may complain of fatigue, restlessness and implicit discomfort in the right upper abdomen. Although rare, mild jaundice may occur. Thus, complications of NAFLD typically include liver fibrosis and subsequent cirrhosis. Liver fibrosis is characterized by the accumulation of extracellular matrix that can be qualitatively distinguished from normal liver. If left unchecked, liver fibrosis can progress to cirrhosis (defined by the presence of cystic nodules), liver and organ failure, and death.
In some embodiments, agents that inhibit the activity or expression of DBI are particularly suitable for treating NASH.
In some embodiments, the subject has pancreatitis, which is an inflammatory disease of the exocrine pancreas, ultimately leading to massive necrotic cell death of acinar cells. Although the mechanism that contributes to this pathology is still unclear, a consensus has been reached in the view that autophagy is impaired during the course of the pathology. Acinar cells are characterized by the inability of large autophagosomes to become autophagosomes, mainly due to depletion of lysosomal proteins (i.e., LAMP 2). Furthermore, it has recently been shown that the absence of Ikk α inhibits autophagy flux and promotes the formation of p 62-positive protein aggregates, thereby promoting the onset of disease. Furthermore, in the acute phase of the disease, a selective autophagy process called "enzymatic autophagy" prevents acinar cells from dying by degradation of the harmful activated zymogen particles. The agents of the invention (such as hydroxycitric acid) can be tested for their ability to prime the enzyme autophagy. In addition, these agents, alone or in combination with lysosomal targeting therapies, may be suitable for ameliorating the symptoms of disease by restoring normal autophagy flux.
In some embodiments, the subject has a proteinopathy. Inducing autophagy by use of the agents of the invention may be particularly suitable for treating proteinopathies. Examples of proteinopathies include, but are not limited to, Alzheimer's disease, cerebral beta-amyloid angiopathy, retinal ganglion cell degeneration, prion diseases (e.g., bovine spongiform encephalopathy, Kuru, Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia), tauopathies (e.g., frontotemporal dementia, Alzheimer's disease, progressive supranuclear palsy, cortical basal membrane degeneration, frontotemporal lobar degeneration), frontotemporal lobar degeneration, amyotrophic lateral sclerosis, Huntington's disease, familial British dementia, familial Danish-type dementia, hereditary cerebral hemorrhage with amyloidosis (CADASIL), Alexandrine disease, Seipinopathies, familial amyloidosis neuropathy, senile systemic amyloidosis, serpinophiles, AL amyloidosis, AA amyloidosis, type II diabetes mellitus, type diabetes mellitus, and Alzheimer's disease, Aortic medial amyloidosis, ApoAI amyloidosis, ApoII amyloidosis, ApoAIV amyloidosis, Finish-type familial amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis/myopathy, cataracts, medullary thyroid carcinoma, atrial amyloidosis, pituitary prolactinoma, hereditary punctate corneal dystrophy, licheniform skin amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar protein deposition, odontogenic tumor amyloidosis, seminal vesicle amyloidosis, cystic fibrosis, sickle cell disease, and severe myopathy.
Agents that inhibit the activity or expression of DBI:
in some embodiments, the agent that inhibits DBI activity is an antibody to DBI.
Thus, as used herein, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region, and includes antibody fragments comprising antigen-binding domains such as Fab ', Fab, F (ab')2, single Domain Antibodies (DAB), TandAbs dimers, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, diabodies, triabodies (scFv-Fab fusion proteins, bispecific or trispecific, respectively), sc-diabodies, kappa (lambda) bodies (scFv-CL fusions), BiTE (bispecific T-cell engagers, scFv-scFv tandem to attract T cells), DVD-Ig (double variable domain antibodies, bispecific formats), SIP (small immune proteins, a minibodies), SMIP ("small modular immunopharmaceutical" scFv-dimer Fc-dimer, scFv-scFv dimer, mini-scFv-fusion, DART (ds-stabilized diabody "Dual Affinity targeting"); small antibody mimetics comprising one or more CDRs, and the like. Techniques for making and using various antibody-based constructs and fragments are well known in the artKnown (see Kabat et al, 1991, which is specifically incorporated herein by reference). Specifically, diabodies are further described in EP 404,097 and WO 93/11161; whereas linear antibodies are further described in Zapata et al (1995). Antibodies can be fragmented using conventional techniques. For example, F (ab')2 fragments can be produced by treating an antibody with pepsin. The resulting F (ab ')2 fragments can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab 'and F (ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments may also be synthesized by recombinant techniques or may be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. Such as Beckmanet et al, 2006; holliger&Hudson,2005；Le Gall et al.,2004；Reff&Heard, 2001; reiteret al, 1996; and Young et al, 1995 and enable the generation of effective antibody fragments. In some embodiments, the antibodies of the invention are single chain antibodies. As used herein, the term "single domain antibody" has its ordinary meaning in the art and refers to the single heavy chain variable domain of an antibody of the type that naturally lacks a light chain that can be found in camelid mammals. Such single domain antibodies are also referred to as "nanobodies". For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as EP 0368684, Wardet et al (
In some embodiments, the antibody is directed to a fragment consisting of the amino acid sequence of amino acid residue 43 to amino acid residue 50 (i.e., an octapeptide or OP).
In some embodiments, the antibodies of the invention are chimeric antibodies, typically chimeric mouse/human antibodies. The term "chimeric antibody" refers to a monoclonal antibody comprising a VH domain and a VL domain of an antibody derived from a non-human animal, a CH domain and a CL domain of a human antibody. As the non-human animal, any animal such as a mouse, a rat, a hamster, a rabbit, and the like can be used. In particular, the mouse/human chimeric antibody may comprise the heavy and light chains of the antibody of the invention.
In some embodiments, the antibody is a humanized antibody. As used herein, "humanized" describes antibodies in which some, most, or all of the amino acids outside of the CDR regions are replaced by corresponding amino acids derived from human immunoglobulin molecules. Humanization methods include, but are not limited to, those described in U.S. Pat. nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference.
In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice at most human immunoglobulin heavy and light chain loci. See, for example, U.S. patent nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584 and the references cited therein, the contents of which are incorporated herein by reference.
In some embodiments, the antibody is a neutralizing antibody. As used herein, the term "neutralizing antibody" is an antibody that inhibits, reduces, or completely eliminates DBI activity. Whether an antibody is a neutralizing antibody can be determined by the in vitro assay described in the examples. Typically, neutralizing antibodies of the invention inhibit DBI activity by at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.
In some embodiments, the neutralizing antibodies of the present invention do not mediate antibody-dependent cell-mediated cytotoxicity, and therefore do not comprise an Fc portion that induces antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the neutralizing antibody does not comprise an Fc domain capable of substantially binding to an FcgRIIIA (CD16) polypeptide. In some embodiments, the neutralizing antibody lacks an Fc domain (e.g., lacks a CH2 and/or CH3 domain) or comprises an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the neutralizing antibody consists of a Fab, Fab '-SH, F (ab')2, Fv, diabody, single chain antibody fragment, or multispecific antibody comprising a plurality of different antibody fragments, or comprises a Fab, Fab '-SH, F (ab')2, Fv, diabody, single chain antibody fragment, or multispecific antibody comprising a plurality of different antibody fragments. In some embodiments, the neutralizing antibody is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from the group consisting of amino acid residues may be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or eliminated Complement Dependent Cytotoxicity (CDC). This method is described in further detail in U.S. Pat. No. 6,194,551 to ldusogene et al.
In some embodiments, the agent that inhibits DBI activity is an aptamer to DBI. Aptamers are a class of molecules that represent an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide sequences with the ability to recognize virtually any type of target molecule with high affinity and specificity. Such ligands can be isolated by systematic evolution of the ligands by exponential enrichment (SELEX) of random sequence libraries. Random sequence libraries can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of unique sequence that is ultimately chemically modified. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein (e.g., E.coli thioredoxin A) selected from combinatorial libraries by two hybridization methods (Colas et al, 1996).
In some embodiments, the agent that inhibits DBI expression is an expression inhibitor. "expression inhibitor" refers to a natural or synthetic compound having the biological effect of inhibiting gene expression. In a preferred embodiment of the present invention, the gene expression inhibitor is an siRNA, an endonuclease, an antisense oligonucleotide or a ribozyme.
In some embodiments, the expression inhibitor is an siRNA. Small inhibitory rnas (sirnas) may also be used as expression inhibitors for use in the present invention. DBI gene expression can be reduced by contacting the patient or cell with small double-stranded RNA (dsrna), or a vector or construct that causes the production of small double-stranded RNA, thereby specifically inhibiting DBI gene expression (i.e., RNA interference or RNAi).
In some embodiments, the expression inhibitor is an endonuclease. The term "endonuclease" refers to an enzyme that cleaves phosphodiester bonds within a polynucleotide strand. Some, such as dnase I, cut DNA relatively non-specifically (regardless of sequence), while many are typically referred to as restriction endonucleases or restriction enzymes, cut only at very specific nucleotide sequences. The mechanisms behind endonuclease-based genomic inactivation generally require a first step of DNA single-or double-strand breaks, which can then trigger two different cellular mechanisms of DNA repair that can be used for DNA inactivation: error prone non-homologous end joining (NHEJ) and high fidelity homology mediated repair (HDR). In a specific embodiment, the endonuclease is CRISPR-Cas. As used herein, the term "CRISPR-Cas" has its general meaning in the art and refers to a clustered regularly interspaced short palindromic repeat sequence, which is a prokaryotic DNA fragment containing a short repetitive base sequence. In some embodiments, the endonuclease is CRISPR-cas9, which is from streptococcus pyogenes. CRISPR/Cas9 systems have been described in US 8697359B1 and US 2014/0068797. In some embodiments, the Endonuclease is CRISPR-Cpf1, which is the recently characterized CRISPR ("
In some embodiments, the expression inhibitor is an antisense oligonucleotide. The term "antisense oligonucleotide" refers to an oligonucleotide sequence that is inverted relative to its normal direction of transcription, and thus expresses an RNA transcript that is complementary to a target gene mRNA molecule expressed in a host cell (e.g., it can hybridize to the target gene mRNA molecule by Watson-Crick base pairing). The antisense strand can be constructed in a number of different ways, so long as it is capable of interfering with the expression of the target gene. For example, the antisense strand may be constructed by reversing the coding region (or a portion thereof) of the target gene relative to the normal direction of transcription to allow transcription of its complement (e.g., the RNA encoded by the antisense and sense genes may be complementary). Furthermore, the antisense oligonucleotide strand need not have the same intron or exon pattern as the target gene, and non-coding and coding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression. As used herein, the term "oligonucleotide" refers to a nucleic acid sequence in the 3'-5' or 5'-3' direction, which may be single-stranded or double-stranded. In particular, antisense oligonucleotides used in the context of the present invention may be DNA or RNA. According to the invention, the antisense oligonucleotides of the invention target mRNA encoding DBI (e.g., SEQ ID NO:2) and are capable of reducing the amount of DBI in a cell. As used herein, an oligonucleotide that "targets" an mRNA refers to an oligonucleotide that is capable of specifically binding to the mRNA. That is, the antisense oligonucleotide comprises a sequence that is at least partially complementary, preferably fully complementary, to a region of the mRNA sequence, the complementarity being sufficient to produce specific binding under intracellular conditions. As will be immediately apparent to one skilled in the art, a sequence that is "fully complementary" to a second sequence refers to the reverse complementary counterpart of the second sequence in the form of a DNA molecule or in the form of an RNA molecule. One sequence is "partially complementary" to a second sequence if one or more mismatches are present. Antisense oligonucleotides of the invention that target DBI-encoding mrnas can be designed by basing the sequence of the mRNA (e.g., using bioinformatic tools). For example, the sequence of SEQ ID NO. 2 can be used as a basis for designing nucleic acids targeting DBI-encoding mRNAs. Preferably, the antisense oligonucleotides according to the invention are capable of reducing the amount of DBI in a cell (e.g. in a cancer cell). Methods for determining whether an oligonucleotide is capable of reducing the amount of DBI in a cell are known to those skilled in the art. This can be done, for example, by: DBI protein expression was analyzed by Western blot and by comparing DBI protein expression in the presence and absence of the antisense oligonucleotides to be tested. In some embodiments, the antisense oligonucleotides of the invention are 12-50 nucleotides in length, e.g., 12-35 nucleotides, 12-30, 12-25, 12-22, 15-35, 15-30, 15-25, 15-22, 18-22, or about 19, 20, or 21 nucleotides in length. For example, the antisense oligonucleotide according to the invention may comprise or consist of 12 to 50 consecutive nucleotides, such as 12 to 35, 12 to 30, 12 to 25, 12 to 22, 15 to 35, 15 to 30, 15 to 25, 15 to 22, 18 to 22 or about 19, 20 or 21 consecutive nucleotides of a sequence complementary to the mRNA of SEQ ID NO. 2. In some embodiments, the antisense oligonucleotides of the invention are further modified, preferably chemically, to increase the stability and/or therapeutic efficacy of the antisense oligonucleotides in vivo. In particular, antisense oligonucleotides used in the context of the present invention may comprise modified nucleotides. Chemical modification can occur at three different sites: (i) on the phosphate group, (ii) on the sugar moiety, and/or (iii) on the entire backbone structure of the antisense oligonucleotide. For example, antisense oligonucleotides can be used as phosphorothioate derivatives with enhanced resistance to nuclease digestion (replacement of non-bridged phosphoryl oxygen atoms with sulfur atoms). 2' -Methoxyethyl (MOE) modifications (such as the modified backbone commercialized by ISIS Pharmaceuticals) are also effective. Additionally or alternatively, the antisense oligonucleotides of the invention may comprise, in whole, in part, or in combination, modified nucleotides which are derivatives having a substitution at the 2' position of the sugar, in particular derivatives having the following chemical modifications: o-methyl (2' -O-Me) substitution, 2-methoxyethyl (2' -O-MOE) substitution, fluoro (2' -fluoro) substitution, chloro (2' -Cl) substitution, bromo (2' -Br) substitution, cyanide group (2' -CN) substitution, trifluoromethyl (2' -CF3) substitution, OCF3 group (2' -OCF3) substitution, OCN group (2' -OCN) substitution, O-alkyl (2' -O-alkyl) substitution, S-alkyl (2' -S-alkyl) substitution, N-alkyl (2' -N-alkyl) substitution, O-alkenyl (2' -O-alkenyl) substitution, S-alkenyl (2' -S-alkenyl) substitution, N-alkenyl (2' -N-alkenyl) substitution, N-alkyl (2' -O-alkenyl) substitution, N-alkenyl (2' -O-alkenyl) substitution, N, SOCH3 radical (2'-SOCH3) substituted, SO2CH3 radical (2' -SO2CH3) substituted, ONO2 radical (2'-ONO2) substituted, NO2 radical (2' -NO2) substituted, N3 radical (2'-N3) substituted and/or NH2 radical (2' -NH2) substituted. Additionally or alternatively, the antisense oligonucleotides of the invention may comprise fully or partially modified nucleotides in which the ribose moiety is used to generate Locked Nucleic Acids (LNAs) in which a covalent bridge is formed between the 2' oxygen and the 4' carbon of the ribose, fixing it in the 3' -endo configuration. These constructs are extremely stable in biological media, capable of activating RNase H and forming a tight hybrid with complementary RNA and DNA. Thus, in a preferred embodiment, the antisense oligonucleotides used in the context of the present invention comprise modified nucleotides selected from the group consisting of: LNA, 2' -OMe analog, 2' -phosphorothioate analog, 2' -fluoro analog, 2' -Cl analog, 2' -Br analog, 2' -CN analog, 2' -CF3 analog, 2' -OCF3 analog, 2' -OCN analog, 2' -O-alkyl analog, 2' -S-alkyl analog, 2' -N-alkyl analog, 2' -O-alkenyl analog, 2' -S-alkenyl analog, 2' -N-alkenyl analog, 2' -SOCH3 analog, 2' -SO2CH3 analog, 2' -NO2 analog, 2' -NO2 analog, 2' -N3 analog, 2' -NH2 analog and combinations thereof. More preferably, the modified nucleotide is selected from the group consisting of: LNA, 2' -OMe analogue, 2' -phosphorothioate analogue and 2' -fluoro analogue. In some embodiments, the antisense is a tricyclo-DNA antisense. The term "tricyclo-DNA (tc-DNA)" refers to a class of constrained oligodeoxyribonucleotide analogues in which each nucleotide is modified by the introduction of a cyclopropane ring to limit the conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle γ (Ittig D, et al, Nucleic Acids Res,2004,32: 346-. Specifically, the additional ethylene bridge between the C (3 ') and C (5') centers of the tc-DNA nucleosides is structurally distinct from DNA, with cyclopropane units fused to the centers to further enhance structural rigidity. See, for example, WO2010115993 for examples of tricyclo-DNA (tc-DNA) antisense oligonucleotides. An advantage of tricyclo-DNA chemistry is that the structural properties of its backbone allow for shortening of the length of the AON while maintaining high affinity and highly specific hybridization to complementary nucleotide sequences. Unexpectedly, tc-DNAAON can be advantageously used in vivo at microgram doses using intramuscular administration, which is at least ten times less than the dose required by conventional antisense oligonucleotide technology. In addition, tc-DNA retains intact activity, with antisense length reduced. Thus, for example, a 13-15 nucleotide tc-DNA AON is highly effective in the ex vivo and in vivo applications exemplified by the present disclosure.
In some embodiments, the agent that inhibits DBI activity consists in a vaccine composition suitable for eliciting neutralizing autoantibodies against DBI when administered to a subject. For the purposes of the present invention, the term "vaccine composition" refers to a composition that can be administered to a human or animal to induce an immune system response; this immune system response results in the production of antibodies against DBI. Typically, the vaccine composition comprises at least one antigen derived from DBI. As used herein, the term "antigen" refers to a molecule capable of specific binding by an antibody or T Cell Receptor (TCR) processed and presented by MHC molecules. As used herein, the term "antigen" also encompasses T cell epitopes. The antigen is additionally capable of being recognized by the immune system and/or capable of inducing a humoral immune response and/or a cellular immune response, leading to the activation of B-and/or T-lymphocytes. An antigen may have one or more epitopes or antigenic sites (B-and T-epitopes). In some embodiments, the antigen is a polypeptide comprising an amino acid sequence (e.g., an epitope) that is at least 80% identical to the sequence of SEQ ID NO. 1 or a fragment thereof. In some embodiments, the antigen is in a polypeptide comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO. 1, or ii) an amino acid sequence having at least 80% identity to the amino acid sequence from amino acid residue 17 to
In some embodiments, the vaccine compositions of the invention comprise an adjuvant. The term "adjuvant" may be a compound that lacks significant activity when administered alone, but can enhance the activity of another therapeutic agent. In some embodiments, the adjuvant is Incomplete Freund's Adjuvant (IFA) or other oil-based adjuvant, which is present in a weight (w/w) ratio of 30-70%, preferably 40-60%, more preferably 45-55%. In some embodiments, the vaccine composition of the invention comprises at least one Toll-like receptor (TLR) agonist selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists. Other adjuvants include cytokines, such as interleukins (II)IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF) and Tumor Necrosis Factor (TNF). In a particular embodiment, the adjuvant is an emulsion with adjuvant properties. Such emulsions include oil-in-water emulsions. Freund's incomplete adjuvant (IFA) is such an adjuvant. Another suitable oil-in-water emulsion is MF59TMAdjuvant comprising squalene, polyoxyethylene sorbitan monooleate (also known as Tween)TM80 surfactant) and sorbitan trioleate. Squalene is a natural organic compound originally obtained from shark liver oil, although it may also be obtained from vegetable sources (mainly vegetable oils), including amaranth seed, rice bran, wheat germ and olive. Other suitable adjuvants are MontanideTMAdjuvants (Seppic Inc., Fairfield N.J.), including MontanideTMISA 50V, a mineral oil-based adjuvant; montanideTMISA 206 and MontanideTMIMS 1312. Although mineral oil may be present in the co-adjuvant, in some embodiments, the oil component of the compositions described herein are all metabolizable oils.
The pharmaceutical composition comprises:
an agent that modulates (i.e., promotes or inhibits) the activity or expression of DBI is administered to a subject in the form of a pharmaceutical composition. Typically, the agents of the invention may be combined with a pharmaceutically acceptable excipient and optionally a polymer of a sustained release matrix (such as biodegradable) to form a therapeutic composition. "pharmaceutically" or "pharmaceutically acceptable" refers to a molecular entity or composition that does not produce an adverse, allergic, or other untoward reaction when administered to a mammal, preferably a human, as the case may be. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. In the pharmaceutical compositions of the invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient alone or in combination with another active ingredient may be administered to a subject in unit administration form, e.g. as a mixture with conventional pharmaceutical carriers. Suitable unit administration forms include oral route forms (such as tablets, gel capsules, powders, granules and oral suspensions or solutions), sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal administration forms, and rectal administration forms. Typically, the pharmaceutical composition contains a medium that is pharmaceutically acceptable for a formulation capable of injection. In particular, these may be isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride, etc. or mixtures of such salts), or dry (especially lyophilized) compositions which allow injectable solutions to be formulated, when sterile water or physiological saline is added as appropriate. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol solutions; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injection is possible. It must be stable under the conditions of preparation and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Solutions containing a compound of the invention as a free base or a pharmaceutically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The agents of the invention may be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide), and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, and the like). For example, the carrier can also be a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. It is also contemplated to prepare more or highly concentrated solutions for direct injection, where it is envisaged that the use of DMSO as a solvent will result in extremely rapid penetration, delivering high concentrations of the active agent to small tumor areas. Once formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described above, but drug-releasing capsules and the like may also be employed. For example, for parenteral administration in aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be employed in accordance with the present disclosure will be known to those skilled in the art. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.
The screening method comprises the following steps:
another object of the invention relates to a method of screening for a compound suitable for modulating autophagy comprising: i) providing a candidate compound, ii) determining whether said candidate compound is capable of modulating the activity or expression of DBI, and iii) positively selecting said candidate compound capable of modulating the activity or expression of DBI.
According to one embodiment of the invention, the candidate compound may be selected from a library of previously synthesized compounds, or a library of compounds for which the structure is determined in a database, or from a library of de novo synthesized compounds or natural compounds. The candidate compound may be selected from the group consisting of: (a) a protein or peptide, (b) a nucleic acid and (c) an organic or chemical compound (natural or non-natural). Illustratively, libraries of preselected candidate nucleic acids can be obtained by performing the SELEX method as described in documents US 5,475,096 and US 5,270,163. In some embodiments, the candidate compound is a peptide derived from DBI or a peptidomimetic of DBI.
Testing whether a candidate compound can modulate the activity or expression of DBI can be determined using or routinely altering assays known in the art. For example, the method can comprise contacting a cell expressing DBI with a candidate compound and measuring a DBI-mediated activity, and comparing the cellular response to a standard cellular response. Typically, the standard cellular response is measured in the absence of the candidate compound. A cellular response below the standard indicates that the candidate compound is capable of inhibiting the activity of DBI. Conversely, a cellular response above the norm indicates that the candidate compound is capable of promoting DBI activity. In some embodiments, the present invention provides methods for identifying ligands that specifically bind to a DBI receptor. For example, a cell compartment, such as a membrane or a preparation thereof, may be prepared from a cell expressing a molecule that binds to a DBI receptor. Methods of determining gene expression are also well known in the art and are typically reporter gene assays (e.g., cells expressing a nucleic acid molecule under the DBI gene promoter, or cells expressing a DBI form labeled with a detectable moiety) or any assay used to determine nucleic acid level expression (e.g., RT-PCR).
In some embodiments, candidate compounds that have been positively selected may undergo further selection steps in view of further determining their performance on autophagy (e.g., with the aid of endogenous fluorescent biosensors or exogenous fluorescent probes, such as described in the examples). In some embodiments, candidate compounds that have been positively selected may be subjected to further selection steps in view of further determining their performance in different in vitro or in vivo assays. For example, the ability of a selected compound to modulate blood glucose, modulate food intake, modulate weight gain or loss, modulate fatty acid oxidation, modulate membrane expression of a glucose transporter (e.g., GLUT-1 or GLUT-4), modulate expression of PPARG, modulate glucose intake (e.g., uptake of a nonradioactive or radioactive glucose isotope), modulate glycolysis, or modulate lipogenesis is determined. Typically, such assays are described in the examples.
Biomarkers:
another object of the invention relates to a method of determining whether a subject is at risk for weight regulation, comprising: i) determining the level of DBI in a blood sample obtained from the subject, ii) comparing the level determined in step i) with a predetermined reference value, and iii) concluding that the subject is at risk of weight regulation when a difference between the level determined in step i) and the predetermined reference value is determined.
In some embodiments, the method of the invention is particularly suitable for determining whether said subject is at risk for weight gain when the level determined in step i) is above a predetermined reference value. In some embodiments, the method of the invention is particularly suitable for determining whether said subject is at risk for weight loss when the level determined in step i) is below a predetermined reference value.
The methods of the invention are particularly suitable for determining whether a subject achieves a response via a diet or drug suitable for modulating weight gain or loss. In some embodiments, the methods of the invention are particularly suitable for determining the risk of relapse.
As used herein, the term "blood sample" refers to any blood sample derived from a patient that contains DBI. In some embodiments, the blood sample is a serum or plasma sample susceptible to contain DBI.
For example, the level is determined by any conventional method for determining the level of a protein in a sample. In some embodiments, the methods of the invention comprise contacting a blood sample with a binding partner capable of selectively interacting with a protein susceptible to being present in the blood sample. The binding partner may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody. In another embodiment, the binding partner may be an aptamer. The polyclonal antibody or a fragment thereof of the present invention can be produced according to a known method by administering an appropriate antigen or epitope to a host animal selected from, for example, pigs, cows, horses, rabbits, goats, sheep, and mice. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in the practice of the present invention may be polyclonal, monoclonal antibodies are preferred. The monoclonal antibodies or fragments thereof of the invention can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for preparation and isolation include, but are not limited to, hybridoma technology. In some embodiments, the binding partner may be an aptamer. Aptamers are a class of molecules that represent an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide sequences that have the ability to recognize virtually any type of target molecule with high affinity and specificity. Binding partners of the invention, such as antibodies or aptamers, may be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any other label known in the art. Labels that typically provide a signal (directly or indirectly) are known in the art. As used herein, the term "labeled" with respect to an antibody is intended to encompass direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance such as a radioactive agent or a fluorophore (e.g., Fluorescein Isothiocyanate (FITC) or Phycoerythrin (PE) or indocyanine (Cy5)), as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. The antibody or aptamer of the invention may be labeled with a radioactive molecule by any method known in the art. The aforementioned assays typically involve the binding of a binding partner (i.e., an antibody or aptamer) to a solid support. Solid supports useful in the practice of the present invention include substrates such as nitrocellulose (e.g., in the form of a membrane or microtiter well); polyvinyl chloride (e.g., flakes or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotizing the paper; a nylon membrane; activating the beads; magnetically responsive magnetic beads, and the like. The level of the biomarker protein may be measured by using standard immunodiagnostic techniques, including immunoassays, such as competition, direct reaction or sandwich-type assays. These assays include, but are not limited to, agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISA; biotin/avidin type assays; performing radioimmunoassay; performing immunoelectrophoresis; and (4) performing immunoprecipitation. More particularly, an ELISA method may be used, wherein e.g. the wells of a microtiter plate are coated with a set of antibodies recognizing the biomarker proteins. A blood sample containing or suspected of containing the biomarker protein is then added to the coated wells. After an incubation period sufficient to allow formation of the antibody-antigen complex, the plate may be washed to remove unbound moieties and detectably labeled secondary binding molecules added. The secondary binding molecules are reacted with any captured sample marker proteins, the plate is washed, and the presence of the secondary binding molecules is detected using methods well known in the art. In some embodiments, the immunoassay may involve the use of 2 antibodies specific for the protein. Typically, a first antibody is used to "detect" the protein and a second antibody is used to "capture" the protein. In some embodiments, the method is achieved by: i) providing the solid support coating with an amount of a first antibody specific for the protein, ii) contacting the sample with the solid support, iii) and adding an amount of a second antibody conjugated to a tag. Measuring the amount of binding partner specifically bound to the tag reveals the amount of protein present in the sample. Typically, the first antibody is directed against an epitope that does not prevent interaction with the second antibody. Typically, a washing step (using any suitable buffer, such as PBS with or without a non-ionic detergent) is performed after steps ii) and iii). Typically, the blocking step is performed with a buffer containing BSA or milk and/or serum (goat or cow) to block non-specific binding of proteins. Measuring the level of biomarker protein (with or without immunoassay-based methods) may also include isolation of compounds: centrifugation based on the molecular weight of the compound; mass and charge based electrophoresis; hydrophobic based HPLC; size exclusion chromatography based on size; solid phase affinity based on the affinity of the compound for the particular solid phase used. Once isolated, the biomarker proteins can be identified based on known "isolation characteristics" (e.g., retention time for the compound) and measured using standard techniques. Alternatively, the protein of interest (e.g., DBI) can be detected and measured by, for example, a mass spectrometer.
In some embodiments, the predetermined reference value is a threshold or cutoff value. Typically, the "threshold" or "cutoff value" may be determined experimentally, empirically, or theoretically. As one of ordinary skill in the art will recognize, the threshold may also be arbitrarily selected based on existing experimental and/or clinical conditions. For example, retrospective measurements of the expression level of DBI in a suitably stored historical patient sample may be used to establish the predetermined reference value. In some embodiments, the predetermined reference value is derived from the DBI level in a control sample from one or more substantially healthy subjects (i.e., normal BMI as defined above). Predetermined reference values must be determined in order to obtain optimal sensitivity and specificity in terms of test function and benefit/risk balance (clinical outcome of false positives and false negatives). Typically, optimal sensitivity and specificity (and threshold) can be determined using Receiver Operating Characteristic (ROC) curves based on experimental data. For example, after determining the levels of a marker in a set of references, the measured levels of the marker in the test sample can be statistically processed using algorithmic analysis to obtain classification criteria that are significant for classifying the sample. The ROC curve is collectively referred to as the receiver operator characteristic curve, also known as the receiver operating characteristic curve. It is mainly used for clinical biochemical diagnosis and test. The ROC curve is a comprehensive indicator of continuous variables reflecting true positive rate (sensitivity) and false positive rate (1-specificity). Which reveals the relationship between sensitivity and specificity by an image synthesis method. A series of different cut-off values (threshold or cut-off, boundary between normal and abnormal results of a diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then, the sensitivity was used as a vertical coordinate and the specificity was used as a horizontal coordinate to plot a curve. The higher the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the upper left corner of the graph is the critical point with both high sensitivity and high specificity values. The AUC value of the ROC curve is 1.0-0.5. When AUC >0.5, the diagnostic results will get better as AUC approaches 1. When the AUC is 0.5-0.7, the accuracy is low. When AUC is 0.7-0.9, accuracy is moderate. When AUC is higher than 0.9, the accuracy is very high. The algorithmic method is preferably performed by a computer. The ROC curve can be plotted using existing software or systems in the prior art, such as: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCP.SAS, DESIGNOC.FOR, MULTIIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0(Dynamic Microsystems, Inc. silver Spring, Md., USA), and the like.
The invention will be further illustrated by the following figures and examples. These examples and drawings, however, should not be construed as limiting the scope of the invention in any way.
Drawings
Figure 1.DBI effect and DBI inhibitory effect on autophagy in humans and mice. (A-D). The effect of extracellular DBI on autophagy of cultured cells. Within the last 2H, H4-GFP-LC3 cells were cultured for 6H (a) in the presence of neutralizing DBI antibody (which is optionally heat inactivated) in the absence or presence of
Figure 2 plasma DBI concentrations in anorexia or obesity patients. Plasma DBI was measured in a cohort of anorexia nervosa (a), obesity (B) or obesity (C) patients one year before or after bariatric surgery, compared to age and gender matched normal weight controls (a, B). Results are mean ± SEM. P <0.001(Student t-test). The results are the average of 5 mice per group. P <0.05, P <0.01, P < 0.001. The complete data is shown in table S1.
Figure 3 glycolysis and feeding-causing effects of DBI. (A-C). Hydrodynamic injection of DBI encoded vectors. Mice (n ═ 5 per group) received i.v. injections of vector only or constructs expressing mouse Dbi cDNA and were monitored over time for blood glucose (a), food intake (B) or weight gain (C). Results are mean ± SEM. P <0.05, P <0.01, P <0.001 for comparison to vehicle-only control. (D-H). The effect of recombinant (rec.) DBI proteins on systemic metabolism. Mice (n ═ 5 per group) were injected intravenously with vehicle alone or rec DBI or DBI-derived peptides TTN or ODN and blood glucose (D), while food intake (E, F) and weight gain (G) were measured at the indicated time points. Alternatively, rec.dbi protein is administered as indicated by the arrow and fatty acid oxidation (H) is measured by respirometry within 24 hours. P <0.05, P <0.01, P <0.001 for comparison to vehicle controls.
FIG. 4 neutralization of the anorexia of extracellular DBI with specific antibodies. (A-D). The effect of DBI neutralization on glucose and feeding behavior. Plasma glucose levels were measured in mice fed or not fed (24 hours) with (each group n-5) 30 minutes after injection of monoclonal anti-DBI (anti-DBI mAb7A) (a) or polyclonal anti-DBI (anti-DBI, ab16871) (C) and isotype control antibodies. Food intake after refeeding was monitored over time (B, D). Results are expressed as mean ± SEM (n ═ 5). P <0.05, # P <0.001, representing anti-DBI effect compared to isotype control, and $ P <0.05, $ $ P <0.001, representing anti-DBI effect in unfed mice compared to unfed control mice.
Figure 5 overall inhibition of DBI neutralization induced liver adipogenesis. The effect of DBI neutralization on various liver protein expression was measured by immunoblotting, with each lane representing one mouse (a). Quantification (B) is the mean ± SEM (n-5) × p <0.05 compared to fed mice receiving the isotype.
Figure 6 anorexia of autoimmunity against DBI. Mice were injected with Keyhole Limpet Hemocyanin (KLH) alone or KLH conjugated with rec.dbi protein (KLH-DBI). Mice that produced IgG autoantibodies against DBI were compared to KLH-only immunized controls to monitor weight loss under starvation conditions (5 animals per group) (a), food intake 24h after starvation (B), and to measure the cumulative weight gain of normal (C) or high fat diet (D) mice (7-8 per group). P <0.05, P <0.01, P <0.001 for the action of DBI-specific autoantibodies.
Detailed Description
Materials and methods
Chemicals, cell lines and culture conditions. Unless otherwise indicated, media and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, CA, USA), plastic from Corning B.V.Life Sciences (Schiphol-Rijk, The Netherlands) and chemicals from Sigma-Aldrich (St Louis, MO, USA). All cell lines were incubated at 37 ℃ in 5% CO2In a medium containing 10% fetal calf serum, 100mg/L sodium pyruvate, 10mM HEPES buffer, 100 units/mL sodium penicillin G and 100. mu.g/mL streptomycin sulfate. In addition, cell type specific culture conditions included Dulbecco's Modified Eagle Medium (DMEM) for human cervical cancer HeLa cells and human brain glioma H4 cells and derivatives thereof expressing GFP-
Plasmid transfection and RNA interference in human cell culture. A plasmid encoding DBI cDNA was obtained from OriGene (Rockville, Md., USA). Transient plasmid transfections were performed with the atractene r reagent (Qiagen, Hilden, Germany) and cells were analyzed 24h after transfection, unless otherwise indicated. Cells were cultured in 6-well or 96-well plates and transfected at 50% confluence. siRNA was reverse transfected with RNAi MaxTM transfection reagent (Invitrogen, Eugene, USA) in the presence of 100nM siRNA specific for DBI and TSPO (Qiagen), and scrambled siRNA was used as a control. siRNA mediated protein down-regulation was controlled by immunoblotting.
Immunofluorescence. Cells were fixed with 4% PFA for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min. Non-specific binding sites were blocked with 5% bovine serum in PBS, followed by staining with primary antibody overnight at 4 ℃. Cells were stained to detect dbi (santa cruz). With appropriate AlexaFluorTMConjugate (Molecular Probes-Invitrogen) developed primary antibody. Nuclei were labeled with Hoechst 33342 (10. mu.g/ml). Standard and confocal fluorescence microscopy evaluations (40X) were performed on IRE2 microscopes (Leica Microsystems) equipped with a DC300F camera and LSM 510 microscope (Carl Zeiss, Jena, Germany) or Leica SPE confocal microscope, respectively. For quantification of the spot mean area, images were captured with a confocal microscope using a 40X objective. The acquired images were converted to 8-bit binary files and the area of a single GFP-LC3 stain with an area greater than four pixels per image was calculated by ImageJ software (NIH). Each experiment was performed at least 3 times, and 40-50 cells were quantified under each condition.
An automated microscope. H4, Hep G2 or HeLa cells stably expressing GFP-LC3 were seeded in 96-well imaging plates (BD Falcon, Sparks, USA) 24H before stimulation. Cells were treated with indicated reagents for 4-6 h. Subsequently, cells were fixed with 4% PFA and counterstained with 10 μ M Hoechst 33342. Images were acquired using a BDpathway 855 automated microscope (BD Imaging Systems, San Jos é, USA) equipped with a 40X objective (Olympus, Center Valley, USA) connected to an automated Twister II plate handler (calipers life Sciences, Hopkinton, USA). Images were analyzed by BDAttovision software (BD Imaging Systems) to determine the presence of GFP-LC3 stain in the cytoplasm. The cell surface was segmented and divided into cytoplasmic and nuclear regions according to standard procedures of the manufacturer. Identification of cytoplasmic GFP-GALT, G Using RB 2x2 and Marr-Hildreth AlgorithmFP-LC3, RFP-FYVE, GFP-GALT-RFP-LC3 and GFP-RFP-LC3 positive spots. Statistical analysis in R software (http://www.r-project.org/) The above process is carried out.
Immunoblotting. For immunoblotting, 25. mu.g of protein were separated on 4-12% Bis-Tris acrylamide (Invitrogen) or 12% Tris-glycine SDS-PAGE precast gels (Biorad, Hercules, USA) and then electrotransferred to ImmobilonTMMembranes (Millipore Corporation, Billerica, USA). The membrane is then cut into different sections depending on the molecular weight of the protein of interest to allow simultaneous detection of different antigens in the same experiment. The membranes were incubated for 1h in 0.05% Tween 20 (v: v in TBS) supplemented with 5% skimmed milk powder (w: v in TBS) and then with primary antibodies specific for DBI (XXX), SQSTM1/p62(Santa Cruz Biotechnology, CA, USA), LC3, FASN, p-p70s6k, p70s6k, p-SREBP, SRP, GLUT1, GLUT4, TSPO, PPARG (Cell Signalling, Danvers, MA, USA) overnight. Visualization was performed using an appropriate horseradish peroxidase (HRP) labeled secondary antibody (Southern Biotech, Birmingham, USA) plus SuperSignal West Pico chemiluminescent substrate (Thermo Scientific-Pierce). Anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH; Chemicon International, Temecula, USA) or anti-actin (Abcam, Cambridge, MA, USA) was used to control equal loading of the lanes.
Mouse experiments and tissue treatment. Wild Type (WT) (Charles River Laboratory, Lentily, France), GFP-LC 3-transgene (gift from professor N.Mizushima), Beclin+/-C57BL/6 (gift of doctor B. Levine), Ambragt/gt(gift of doctor Boya, P.), Atg4b-/-C57BL/6 mice (gift of Dr. Lopez-Otin) and maintained according to FELASA and guidelines of the ethical committee for animal experiments (CE n.26:2012-65, 2012-67; Val de Marne, France). Mice were housed in a temperature-controlled environment with 12h light/dark cycles and received food and water or a High Fat Diet (HFD) ad libitum. Mice were starved for 24-48h, or injected intraperitoneally or intravenously with DBI, DBI-derived peptides or DBI-specific antibodies and sacrificed after 1h to 6 h. Immediately following extraction, the tissues were frozen in liquid nitrogen and buffered in 20mM Tris using a
And (3) DBI detection: after in vivo treatment, plasma from the blood collection tubes was collected by centrifugation at 15000rpm for 30 minutes and the amount of DBI in the plasma was determined using DBI ELISA (Mybiosource MBS2025156) according to the manufacturer's instructions. For in vitro experiments, H4, Hep G2 or HeLa cells were seeded 24H before stimulation in 96-well imaging plates (BD Falcon, Sparks, USA). Cells were treated with indicator for a specified time and supernatants were collected and the amount of DBI in the supernatants was determined using DBI ELISA (abnova 0532 DBI (human) ELISA).
And (4) immunization. 6-8 week old C57BL/6 male mice obtained from Harlan France (Gannat, France) were immunized subcutaneously on the base of the tail with 100. mu.g of alum-precipitated KLH (Calbiochem, La Jolla, Calif.) in 100. mu.l of balanced salt solution. DBI-KLH was prepared by cross-linking DBI with Keyhole Limpet Hemocyanin (KLH). Transgenic mice expressing DBI autoantibodies received saline KLH-DBI emulsified in Montanide ISA51vg adjuvant by intramuscular injection (30g, 10g once a week for 4 weeks). To produce KLH-DBI conjugates, murine DBI was mixed at a molar ratio of 1:20 and gradually adjusted to 0.25% final (v/v) glutaraldehyde. The reaction was stopped by addition of glycine solution. After ultrafiltration (Millipore; Billerica, Massachusetts, USA), a formaldehyde solution was added to a final concentration of 0.2% (v/v). The reaction was quenched by addition of glycine solution and then subjected to ultrafiltration using a 100kDa membrane with 70mM phosphate buffer (pH 7.8). DBI-KLH was stored at 4 ℃. IFNgf for use as a control antigen was prepared in the same manner except that the crosslinking reaction was carried out in the absence of KLH and the final membrane had a molecular weight cut-off of 10 kDa. Protein concentration was determined by Bradford assay.
A nematode strain: we followed standard procedures to maintain the caenorhabditis elegans (c. For all our experiments, the feeding temperature was set at 20 ℃. We used DA2123: WT; is [ p ]lgg-1GFP::LGG-1+rol-6(su1006)],MAH14:daf-2(e1370)；[plgg-1GFP::LGG-1+rol-6(su1006)]And MAH28: aak-2(ok 524); [ p ]lgg-1GFP::LGG-1+rol-6(su1006)]For assessment of autophagy (40, 41). The first strain was associated with SV62: acbp-1(SV62) I and quadruple FE0017: acbp-1(SV62) I; acbp-6(tm2995) II; acbp-4(tm2896) III; the acbp-3(sv73) X strains (42) were hybridized to monitor autophagy after exhaustion of the acbp family gene. For pharyngeal pumping measurements, the SV62 and FE0017 strains were used in combination with DA465: eat-2(ad465) II, a genetic model for reducing pharyngeal pumping.
Autophagy measurements of caenorhabditis elegans: autophagy was measured according to the method described in (43). In short, ten well-reared adult worms with their respective genetic backgrounds were allowed to lay eggs on NGM or RNAi plates. After four hours, the parents were removed and the plates were placed at 20 ℃. Two days later, the synchronized animals were collected, anesthetized with 10mM levamisole and mounted on glass slides for microscopic examination. The number of GFP:LGG-1 positive autophagy plaques was measured in subcutaneous seam cells at the larval stage of L3-L4 (44). Pharyngeal pumping was measured as described in (45). The grinder motion of a freely moving animal was measured under a stereomicroscope. Three independent measurements were made for each individual and the average number of pumps per animal was recorded. Starvation was achieved by placing the animals in bacteria-free NGM plates for 24 hours. Prior to observation, animals were placed on plates inoculated with OP50 for half an hour recovery.
Immunohistochemistry of mouse brain. Mice were deeply anesthetized with pentobarbital (Nembutal, Abbott Laboratories, Chicago, Ill.; 80mg/kg ip) and intracardially perfused with phosphate buffer (PB; 0.1M) followed by 4% paraformaldehyde (in 0.1M PB). Brains were removed, fixed in the same fixative for 2h, cryoprotected in 20% sucrose solution (in 0.1M PB) for 48h, and then in CO2And (5) quick-freezing. Coronal sections (20 μm) were cut in a cryostat (CM 3050Leica, Nussloc, Germany). Hypothalamic sections were collected in three separate series and mounted on microscope slides for thawingOn a chip (SuperFrost Plus, Faust, Schaffhausen, Switzerland). After air drying at room temperature and rehydration in PBS, sections were incubated in blocking solution for 2h (1.5% rabbit normal serum + avidin; Vector Laboratories, Burlingame, Calif.). Primary antibody (polyclonal goat anti-c-Fos, Santa Cruz; 1:10,000+ biotin, vector laboratories) was administered at 4 ℃ for 48 h. Unbound antibody was removed by washing in PBS and the sections were incubated with a secondary antibody (biotinylated rabbit anti-goat, Vector-Elite ABC Kit, Vector Laboratories; 1:200) for 2h at room temperature. Diaminobenzdiene (DAB) was used as chromogen [ containing 0.02% H in [ ] after incubation in ABC solution (Vectastain-Elite ABC Kit, Vector Laboratories)2O20.04% in PBS and for color enhancement 0.08% NiCl2(×6H2O),0.01％CoCl2(×6H2O)]. Finally, sections were dehydrated in fractionated alcohol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany).
Measurement of yeast autophagy: autophagy was monitored BY vacuolar localization of Atg8p BY cells expressing the EGFP-Atg8 fusion protein using fluorescence microscopy or BY alkaline phosphatase (ALP) activity using BY4741 wild type or dbi1 transformed cells according to the disclosed method.
Results and discussion
Autophagy ("autophagy") constitutes one of the most dramatic, although finely regulated, phenomena in cell biology and plays a key role in maintaining cellular and biological homeostasis by promoting the renewal of cytoplasmic structures and allowing cells to adapt to changing and stressful conditions, including nutritional deficiencies (1, 2). Several cell secretions free of leader proteins, which can only be released by non-canonical pathways bypassing the golgi, are closely associated with autophagy (3-7). One such protein is the phylogenetic ancient factor known as diazepam binding protein (DBI) or acyl-coa (coa) binding protein (ACBP) (3, 4). Human or mouse DBI is a small 87 amino acid (10kDa) protein with two completely different functions, namely as an intracellular ACBP (where it binds to a long-chain acyl CoA molecule) and as an extracellular DBI (where the intact protein or its whole protein isThe cleavage product, triacontatetranuropteride [ TTN, residues 17-50]And octadecaneuropeptide [ ODN, residues 33-50]Can be used for treating gamma-aminobutyric acid (GABA) type A receptorAR) interacts with and modulates its activity as a GTP protein-coupled receptor (GPCR) (8-10). DBI and its proteolytic fragments also bind to Peripheral Benzodiazepine Receptors (PBR) (11-13) as well as unidentified GPCRs (ODN-GPCRs) (14-17). Here we address the question as to whether DBI secretion is likely to be involved in autophagy feedback regulation.
Human cell lines cultured without Nutrition (NF) or treated with Rapamycin (RAPA) under autophagy-stimulating conditions showed reduced expression of DBI in cells, which could be inhibited by addition of lysosomal inhibitors such as paflunomide a1(BAFA1), chloroquine, and hydroxychloroquine, and by deletion of the essential autophagy gene/protein ATG5 (data not shown). Under baseline conditions, soluble DBI could be detected in culture supernatants, but increased in NF cultures unless BAFA1 was added or ATG5 was removed (data not shown). Similarly, autophagy competent wild-type (WT) mice that suffered from starvation for 24h (rather than autophagy-deficient Becn 1)+/-) The intracellular content of DBI in several organs was decreased (data not shown), and it is known that starvation for 24h induced autophagy in most cells of the body (18). At the same time, Becn1 from WT (but not from a partial autophagy defect)+/-、Atg4b-/-And Ambra1gt/gtMice) had elevated DBI levels following plasma starvation (data not shown). These results demonstrate that in vivo autophagy induction causes intracellular DBI release into extracellular compartments.
Depletion of DBI by small interfering rna (sirna) reduced NF-stimulated autophagy in cultured human cells (data not shown), while overexpression stimulated autophagy flux (data not shown). This result was obtained when autophagy was monitored by following redistribution of microtubule-associated protein 1A/1B light chain 3B (LC3) coupled to Green Fluorescent Protein (GFP) to autophagosomes and by measuring LC3 lipidation (resulting in an increase in its electrophoretic mobility) (data not shown). At the same time, e.g. MTOR substrate p70S6KIncreased phosphorylation indicates that DBI silencing increases kinase activity of a mechanical target of rapamycin (MTOR) (negative regulator of autophagy) (data not shown)Display). Thus, intracellular DBI that intersects the MTOR pathway can negatively regulate MTOR and positively regulate autophagy through direct molecular interaction (19) with late endosomal/lysosomal aptamers, MAPK, and MTOR activator 5(LAMTOR 5). Autophagy-dependent DBI depletion from cells can activate autocrine feedback loops, leading to self-restriction of the autophagy process.
Knock-out of the yeast (s.cerevisiae) acb1 gene, which encodes the DBI ortholog, inhibited NF-induced autophagy (data not shown), while knock-out of the nematode (caenorhabditis elegans) acbp-1 gene alone, or of the nematode (caenorhabditis elegans) acbp-1 gene and several homologues, which are present in this species, but absent in mammals, stimulated autophagy (data not shown). This difference suggests that this phylogenetically ancient protein may have different autophagy-regulating functions in single and multi-cellular environments. Indeed, when DBI is deficient in siRNA in most cultured human cells mixed with a few cells that still express DBI (which inhibits autophagy in these cells, data not shown), this procedure enhances autophagy in the latter (data not shown). Similarly, the addition of antibodies neutralizing extracellular DBI in culture media stimulated autophagy flux (fig. 1.a, B), while the addition of recombinant (rec) DBI protein (or proteins of its TTN and ODN fragments) inhibited NF-induced autophagy in cultured human cells (fig. 1C-D). Similarly, neutralization of extracellular DBI induced autophagy in various organs by intraperitoneal (i.p.) injection of specific antibodies into mice (fig. 1E), while systemic intravenous (i.v.) or i.p. administration of rec.dbi protein inhibited starvation-induced autophagy (fig. 1F). These results indicate that extracellular DBI inhibits autophagy (in contrast to the fact that intracellular DBI stimulates autophagy), which means that autophagy-induced DBI release from cells may be involved in paracrine feedback loops.
In a cohort of 52 anorexia nervosa patients, the plasma DBI concentration was abnormally low compared to age and gender matched controls of normal Body Mass Index (BMI) (fig. 2A), confirming the previous report (20) for 24 anorexia patients. More importantly, obese individuals had abnormally high DBI concentrations (decreased after bariatric surgery), which were associated with high circulating insulin levels (fig. 2B, C). Similarly, genetically obese Ob/Ob mice, which were leptin receptor deficient, exhibited enhanced circulating DBI levels (data not shown). Based on these findings, we investigated whether DBI might regulate general metabolism. To this end, rec.dbi protein and anti-DBI antibody were injected into fed and starved mice, respectively, and their organs were subjected to mass spectrometric metabolomics analysis two hours later. The dbi protein causes hypoglycemia. In contrast, DBI neutralization reversed hypoglycemia induced by starvation and further exacerbated plasma levels of 2-hydroxybutyrate, the ketone body induced by starvation (data not shown). Thus, we decided to study the effect of DBI on weight control in glucose and fatty acid metabolism.
Hydrodynamic injection of cDNA encoding DBI increased hepatic expression of DBI, resulting in hypoglycemia, increased food intake, and induced weight gain (fig. 3A-C). Similarly, systemic (i.p. or i.v.) injection of rec.dbi protein (or a protein of its peptide fragment TTN or ODN) stimulated hypoglycemia triplet, food intake and weight gain (fig. 3D-G). Meanwhile, rec.dbi protein reduced fatty acid oxidation at systemic level as determined by respirometry (fig. 1H). The finding that DBI protein has a feeding-causing effect is contrary to previous reports (21, 22) showing that administration of DBI fragments to the brain is ineffectual. Therefore, rec.dbi protein injected by i.p. or i.v. route may act through the periphery rather than the central nerve. In fact, systemic administration of rec.dbi rapidly (30 min) resulted in liver upregulation of glucose transporter (GLUT1) and peroxisome proliferator-activated receptor gamma-gamma (PPARG), which upregulated adipogenesis by Fatty Acid Synthase (FASN) (data not shown). Thus, rec.DBI enhancement14Incorporation of C atoms from glucose into visceral fat (data not shown). Furthermore, rec.dbi stimulated basal and maximal glycolysis when added to human HepG2 hepatocytes (data not shown). Reversing DBI-induced hypoglycemia by i.p. injection of glucose prevented hyperphagia (data not shown). Thus, DBI drives glucose uptake, glycolysis, and lipogenesis, eventually causing hypoglycemia, triggering a feeding response.
In view of the feeding-causing effect of rec.dbi protein, we investigated whether depletion or neutralization of endogenous DBI would cause anorexia. Carrying a constitutive Dbi knockout (Dbi)-/-) Is smallMice die (23) or are affected by a variety of defects, including their epidermal barrier function (24-33), apparently cannot be used to distinguish between the intracellular and extracellular functions of DBI. We generated mice that could conditionally knock-out Dbi by tamoxifen injection (using tamoxifen (Tam) induced Cre recombinase mediated excision of floxed Dbi) (data not shown). Tam-induced systemic knockout DBI killed a portion of adult C57Bl/6 mice fed normal diet (data not shown), failed to impair survival of High Fat Diet (HFD) mice (data not shown), but sensitized the mice to starvation-induced death (data not shown). Although glucose levels were maintained within the normal glycemic range (data not shown), DBI-deficient mice gained weight loss due to starvation (data not shown). To neutralize only extracellular DBI, we prepared monoclonal antibodies (mAb 7A, IgG). Systemic (i.p.) injection of different anti-DBI antibodies increased plasma glucose levels in fed and starved mice (fig. 4A, C) and decreased food intake after starvation (fig. 4B, D). Very similar results were obtained with several commercial polyclonal antibodies neutralizing DBI (data not shown). In contrast to systemic DBI gene knock-out, mAb7A and polyclonal antibodies did not cause death even in starvation mice. Blocking of DBI inhibited systemic fatty acid oxidation at baseline (data not shown) and starvation (data not shown). Despite the induction of hyperglycemia by DBI neutralization, starved mice exhibited a decrease in plasma insulin levels, C-peptide, and Gastric Inhibitory Peptide (GIP) (data not shown).
In the liver, neutralizing DBI reduced the expression of PPARG and FASN, as it caused inhibitory phosphorylation of sterol regulatory element binding transcription factor 1(SREBF1), corresponding to inhibition of adipogenesis (fig. 5). Therefore, neutralizing DBI can reduce fatty liver in the context of a fat-fatty diet that is obese.
Next, we investigated the possibility of breaking self-tolerance to DBI and inducing the production of neutralizing autoantibodies by immunizing mice with DBI coupled to Keyhole Limpet Hemocyanin (KLH) and a potent adjuvant (34). Autoantibody spikes that persistently neutralize DBI in circulation (data not shown) had a profound effect on metabolism, although there was no lethal effect (as recorded for systemic knockouts), resulting in increased weight loss during hunger (fig. 6A) and decreased refeeding after hunger (fig. 6B). Furthermore, following autoimmunity against DBI, the weight gain typically found in mice fed normal diet or HFD was reduced (fig. 6C, D). In HFD-fed mice, immunity against DBI down-regulated hepatic lipogenesis stimulating Factor (FASN), increased hepatic carnitine palmitoyl transferase-1 (CPT1, which is essential for fatty acid uptake), increased carnitine fatty acid ether in the liver, inhibited fatty liver, decreased hyperlipidemia with various free fatty acids, and up-regulated
Metabolomic comparisons of different tissues from starved mice and mice subjected to DBI neutralization revealed that Brown Adipose Tissue (BAT) (data not shown) and plasma (data not shown) were more similar than liver and muscle (not shown). Although the effect of DBI neutralization on metabolism must be attributed to peripheral effects (outside the central nervous system), antibody-mediated DBI blockade inhibited neurons in the food-borne hypothalamic lateral region (LH) and activated neurons in the anorexia peritoneum medial nucleus (VMN), as determined by assessing phosphorylation of the transcription factor c-Fos (data not shown). Taken together, these results indicate that both passive and active immunity against DBI exert potent anti-obesity effects.
Our data point to a model in which starvation-induced autophagy undergoes three levels of DBI-mediated feedback regulation. Autophagy results in secretion of DBI, depletion of this autophagy-promoting factor from the cell (autocrine regulation), and then DBI accumulated in the extracellular space acts on other cells to inhibit autophagy (paracrine regulation). In addition, circulating DBI stimulates eating behavior, thus increasing nutrient uptake and removing the main cause of autophagy induction (endocrine regulation). The latter effect appears to be phylogenetically conserved, as caenorhabditis elegans, which experienced depletion of one or several DBI orthologs, showed a decrease in pharyngeal pumping (data not shown). Thus, DBI may be involved in the primary reflex, where nutrient consumption stimulates feeding behavior by inducing autophagy.
In addition to having autophagy-inhibiting effects, extracellular DBI also has potent regulatory effects on systemic metabolism. In adolescents with anorexia nervosa, circulating DBI levels are low. This is in contrast to the short-term starvation-induced increase in DBI levels observed in mice. The reason for this difference remains elusive. However, it is easily speculated that the decrease in DBI levels associated with anorexia (probably due to long-term readjustment of the settings determining the expression of transcriptional levels of DBI) (35) may be related to phenotype, since deletion of the gene encoding DBI or neutralization of the DBI protein has an anorexic effect in mice, reducing food intake after hunger. In sharp contrast, providing extracellular DBI by systemic injection of recombinant proteins (or active peptide fragments thereof) stimulates food intake by favoring hypoglycemia, which in turn up-regulates glucose uptake into hepatocytes and increases glycolysis and lipogenesis. In fact, morbidly obese patients or mice exhibit elevated plasma DBI levels. The reason for the increased DBI expression remains unclear. Obesity is associated with autophagy inhibition (36, 37), meaning that altered autophagy flux may not account for increased circulating DBI. Conversely, obesity-related DBI elevation may promote autophagy inhibition, thereby counteracting weight loss and tending to gain weight (38, 39). In addition, increased extracellular DBI levels are beneficial for the feeding and adipogenic responses, as observed, loss or neutralization of DBI can suppress appetite, reduce weight gain, and inactivate HFD-induced obesity and fatty liver. Neutralization of DBI can be achieved by injection of monoclonal or polyclonal antibodies and induction of autoantibodies. The latter strategy may be particularly useful for preventing or treating morbid obesity if long-term DBI blocking is protected from harmful side effects and constitutes a desired therapeutic goal.
Reference to the literature
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