WO2023083010A1 - 人类受试者心肌病的治疗 - Google Patents

人类受试者心肌病的治疗 Download PDF

Info

Publication number
WO2023083010A1
WO2023083010A1 PCT/CN2022/127868 CN2022127868W WO2023083010A1 WO 2023083010 A1 WO2023083010 A1 WO 2023083010A1 CN 2022127868 W CN2022127868 W CN 2022127868W WO 2023083010 A1 WO2023083010 A1 WO 2023083010A1
Authority
WO
WIPO (PCT)
Prior art keywords
rala
ralgapα1
complex
ralgapα2
pharmaceutical composition
Prior art date
Application number
PCT/CN2022/127868
Other languages
English (en)
French (fr)
Inventor
陈帅
朱桑桑
全超
Original Assignee
南京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京大学 filed Critical 南京大学
Publication of WO2023083010A1 publication Critical patent/WO2023083010A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)

Definitions

  • the present invention relates to the treatment of cardiomyopathy of a human subject, in particular to a method of using the RalGAP ⁇ -RalA signaling model to positively regulate the activity of SERCA2 in cardiomyocytes, thereby providing protection to the heart.
  • Heart Failure Hypertension and its associated cardiovascular and heart diseases are among the leading causes of death worldwide.
  • the pressure overload caused by high blood pressure can cause cardiac dysfunction and lead to heart failure (Heart Failure).
  • the calculated lifetime risk of developing heart failure is expected to increase with age. From less than 2% of those under 60 years of age to more than 10% of those over 75 years of age (Metra M, Teerlink JR, Lancet 2017; 390:1981-1995).
  • persons with high blood pressure are at higher risk of heart failure (Lloyd-Jones DM et al., Circulation 2002; 106:3068–3072).
  • Patients with heart failure have a poor prognosis, with high rates of hospitalization and mortality.
  • the pathological mechanism by which hypertension causes heart failure remains unclear.
  • the circulation of calcium ions (Ca 2+ ) between the sarcoplasmic reticulum (Sarcoplasmic Reticulum, SR) and the cytosol of cardiomyocytes determines the cardiac contractility.
  • Pressure overload affects the circulation of Ca 2+ in the heart and leads to an increase in Ca 2+ in the cytosol, which weakens the contraction force of the heart. Therefore, regardless of the triggering mechanism of heart failure, the abnormal distribution of intracellular Ca 2+ caused by the reduced Ca 2+ uptake of intracellular SR is a fundamental factor (Schwinger RH et al., J Mol Cell Cardiol. 1999; 31( 3):479-91; Bers D et al., Ann NYAcad Sci 2006; 1080:165-177).
  • SERCA2 Sarcoplasmic reticulum/endoplasmic reticulum Ca 2+ ATPase 2
  • SERCA2 is an ATP-dependent Ca 2+ transport pump and a key enzyme that regulates the reuptake of Ca 2+ from the cytosol.
  • Dysfunction of SERCA2 is evident in heart failure. It is known that the function of SERCA2 is positively and negatively regulated by various mechanisms. These regulatory mechanisms include phosphoprotein binding, phosphorylation, and ubiquitination-like modifications. For example, the binding of phosphoproteins can inhibit the activity of SERCA2 and slow down the reuptake of Ca2 + in SR.
  • SERCA2Thr 484 by striated muscle preferentially expressed protein kinase enhanced Ca transport activity.
  • SPEG protein kinase
  • ubiquitination of SERCA2 maintains ATPase activity and stability, which is significantly reduced in failing hearts.
  • Ral-GTPases consisting of RalA and RalB are key regulators of a variety of cellular processes. Although RalA and RalB are somewhat duplicated, they have distinct functions in many processes.
  • the intrinsic activities of RalA and RalB are regulated by their upstream regulator Ral-GTPase activating protein (RalGAP) complex.
  • RalGAP upstream regulator Ral-GTPase activating protein
  • the RalGAP complex switches these two small G proteins, RalA and RalB, from the GTP-bound state to the GDP-bound form.
  • RalGAP complex-1 or RalGAP ⁇ 1 complex, both have the same meaning
  • RalGAP complex-2 or RalGAP ⁇ 2 complex, both have the same meaning
  • the invention relates to a medicine for preventing and treating cardiomyopathy or heart failure.
  • the drug of the present invention can target SERCA2 in cardiomyocytes, and positively regulate the activity of SERCA2; in particular, it can positively regulate SR Ca 2+ reuptake of SERCA2 in cardiomyocytes.
  • the drug of the present invention regulates the intracellular balance of Ca 2+ in cardiomyocytes by regulating the function of SERCA2 by itself or in combination with other drugs.
  • the agents of the invention are capable of up-regulating SERCA2a oligomerization.
  • the medicaments of the present invention are capable of preventing and treating cardiomyopathy or heart failure in human subjects or other vertebrate animals.
  • the drug of the present invention can be a biological product, including: protein, protein complex, polypeptide and antibody.
  • this application relates to the application of a pharmaceutical composition in the preparation of drugs for the prevention or treatment of cardiomyopathy, wherein the pharmaceutical composition may include: RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2/ ⁇ complex RalA in the form of RalA, RalA, GDP, or their splice variants, truncation, fragmentation, substitution, addition or deletion mutants, recombination mutants, motif sequences, fusion proteins and homologues, homologous genes one or more.
  • said cardiomyopathy is characterized by abnormalities in SERCA2-mediated calcium ion transport, such as heart failure, especially chronic heart failure.
  • this application relates to the application of a pharmaceutical composition in the preparation of drugs that positively regulate SERCA2a oligomerization, wherein the pharmaceutical composition includes: RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2/ ⁇ RalA in complex, RalA, GDP-bound form, or their splice variants, truncation, fragment, substitution, addition or deletion mutants, recombination mutants, motif sequences, fusion proteins and homologues, in homologous genes one or more.
  • the present application relates to the application of a pharmaceutical composition in the preparation of drugs that positively regulate SERCA2 activity, wherein the pharmaceutical composition includes: wherein the pharmaceutical composition includes: RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2/ ⁇ complex, RalA, GDP-bound RalA, or their splice variants, truncations, fragments, substitutions, addition or deletion mutants, recombination mutants, motif sequences, fusion proteins and homologs One or more of objects and homologous genes.
  • the invention also provides nucleic acid molecule compositions comprising or encoding the biological products of the invention. These compositions are suitable for pharmaceutical use and administration to patients.
  • the composition generally contains one or more biological products of the invention and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • the compositions of the biological products of the present invention may also contain other active compounds that provide complementary, additional or enhanced therapeutic functions.
  • the biologicals of the invention or nucleic acid molecules encoding the biologicals of the invention may be included in vectors that will protect the biologicals or nucleic acid molecules encoding these biologicals from rapid elimination from the body, and/or Or deliver biological products to designated tissues and organs.
  • the present application relates to a pharmaceutical composition, comprising: a first delivery carrier and one or more of the following active substances: (1) RalGAP ⁇ 1; (2) RalGAP ⁇ 1/ ⁇ complex; (3 ) RalGAP ⁇ 2; (4) RalGAP ⁇ 2/ ⁇ complex; (5) RalA, the GDP-bound form of RalA, or splice variants of (1)-(5), truncation, fragment, substitution, addition or deletion mutants, recombinant One or more of mutants, motif sequences, fusion proteins and homologues, homologous genes.
  • the first delivery vehicle can be, for example, a controlled release formulation, including implants and liposomes, nanoparticles, hydrogels, microspheres, microencapsulated delivery systems, and the like.
  • the first delivery vehicle can use biodegradable, biocompatible polymers, such as polyesters, polyacrylates and their copolymers, such as polylactic acid and its copolymers, poly- ⁇ -hydroxycarboxylates, ethylene Vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, polyacrylic acid polymerized from monomers such as methacrylic acid, acrylate and acrylates such as butyl methacrylate and propylene glycol methacrylate wait.
  • biodegradable, biocompatible polymers such as polyesters, polyacrylates and their copolymers, such as polylactic acid and its copolymers, poly- ⁇ -hydroxycarboxylates, ethylene Vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester
  • the first delivery vehicle is chitosan and other polysaccharides, alginate, collagen, gelatin, fibrin, glycosaminoglycan, agarose, and the like.
  • liposomal suspensions containing biologicals can also be used as pharmaceutically acceptable carriers.
  • the first delivery vehicle is a nanobiomedical vehicle.
  • the present application relates to a pharmaceutical composition, comprising: a second delivery vector and a nucleic acid molecule encoding and capable of expressing one or more of the following active substances in cardiomyocytes: (1) RalGAP ⁇ 1 (2) RalGAP ⁇ 1/ ⁇ complex; (3) RalGAP ⁇ 2; (4) RalGAP ⁇ 2/ ⁇ complex; (5) RalA, GDP-bound form of RalA; or splice variants of (1)-(5), truncated , one or more of fragments, substitutions, addition or deletion mutants, recombination mutants, motif sequences, fusion proteins and homologues, homologous genes.
  • the pharmaceutical composition as described above can introduce the DNA encoding the biological product of the present invention into cells, and then express the biological product of the present invention encoded by the nucleic acid molecule in the cells.
  • delivery of the biologicals of the invention can be achieved using a second delivery vehicle.
  • the second delivery vector is a recombinant expression vector such as a chimeric virus or a colloidal dispersion system or a nanobiomedical vector, and various viral vectors that can be used to introduce DNA into cells include adenovirus, herpes virus, vaccinia virus or RNA viruses such as retroviruses.
  • colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems.
  • Lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vesicles that can be used as delivery vehicles. RNA, DNA, or whole virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form. Methods for the efficient transfer of genes into cells using liposome vectors are known in the art.
  • the composition of liposomes generally includes a combination of phospholipids, typically in combination with a steroid, especially cholesterol, although other phospholipids or lipids may also be used.
  • the biological product of the present invention is RalA S28N recombinant protein or a nucleic acid molecule encoding RalA S28N recombinant protein, which is used to express or overexpress RalA S28N recombinant protein in cardiomyocytes.
  • the biological product of the present invention is an expression cassette for expressing the RalA S28N mutant driven by a Ctnt promoter in an adeno-associated virus (AAV) system.
  • the RalA S28N mutant was specifically expressed in the heart by intravenous injection of the AAV virus used to express the RalA S28N mutant.
  • the invention provides methods and uses of upregulating SERCA2 activity in cardiomyocytes; in particular, methods and uses of upregulating SR Ca2 + reuptake by SERCA2 in cardiomyocytes. Further, in some embodiments, the present invention also provides regulating the intracellular balance of Ca 2+ in cardiomyocytes by regulating the function of SERCA2. In some embodiments, the present invention also provides methods and uses for up-regulating oligomerization of SERCA2a. In some embodiments, the present invention provides methods and uses for the prevention and treatment of cardiomyopathy or heart failure.
  • the present application relates to a method for positively regulating oligomerization of SERCA2a, comprising: administering to a subject an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the present application relates to a method of positively regulating SERCA2 activity, comprising: administering to a subject an effective amount of any of the pharmaceutical compositions described above. In some embodiments, the present application relates to a method for preventing and treating cardiomyopathy in a subject, comprising: administering to the subject an effective amount of any one of the pharmaceutical compositions described above.
  • the present application also relates to a method for identifying substances capable of affecting the RalGAP ⁇ -RalA signaling pathway, comprising: providing polypeptides of proteins in the RalGAP ⁇ -RalA signaling pathway or fragments, derivatives, homologues or mutants thereof; allowing 5'- Contacting said polypeptide or fragment, derivative, homologue or mutant thereof with a test substance under conditions in which ATP, 5'-GTP and/or 5'-GDP binds to said polypeptide; and determining said test substance Whether it binds to said polypeptide or a fragment, derivative, homologue or mutant thereof.
  • the proteins in the RalGAP ⁇ -RalA signaling pathway include RalGAP ⁇ 1, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2, RalGAP ⁇ 2/ ⁇ complex, RalA, GDP-bound RalA, and SERCA2.
  • the above method further includes administering a substance that has been determined to be capable of binding the polypeptide to any polypeptide of the signaling pathway or a fragment, derivative, homologue or mutant thereof.
  • a substance that has been determined to be capable of binding the polypeptide to any polypeptide of the signaling pathway or a fragment, derivative, homologue or mutant thereof have been produced recombinantly.
  • the above method further includes: determining whether the test substance can replace any polypeptide or fragment thereof on the Ral-GDP signaling pathway without affecting the activity of the original signaling pathway.
  • the present application also relates to a method for quantitatively or qualitatively detecting the SERCA2 activity of a subject, comprising: providing a substance interacting with a protein in the RalGAP ⁇ -RalA signaling pathway; subjecting the substance interacting with a protein in the RalGAP ⁇ -RalA signaling pathway to Exposure to cells, body fluids or tissues of the subject; and assessment of protein levels in the RalGAP ⁇ -RalA signaling pathway of the subject.
  • the present invention also provides a diagnostic tool that can be used as a quantitative or qualitative detection of SERCA2 activity.
  • a biological product such as an antigen, that interacts with RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complex and/or the GDP-bound form of RalA is provided.
  • These biologicals can be fluorescently labeled.
  • Samples can be incubated with labeled biologicals, excess unbound protein can be washed away, and the tissue can then be assessed for fluorescent activity, which will indicate the presence of RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complexes and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complex and/or GDP-bound form RalA.
  • biological products that interact with RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complexes and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complexes and/or GDP-bound RalA can be used for detection in cells, body fluids, tissues or organisms Presence and content of RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complex and/or GDP-bound form RalA.
  • the detected amount of RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complex and/or GDP-bound form RalA can be correlated with the activity of SERCA2.
  • the present invention also includes a kit for detecting the level of RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complex and/or GDP-bound form RalA in a sample, comprising at least one a biological product, such as an antigen, whether labeled or unlabeled, that interacts with RalGAP ⁇ 1 and/or RalGAP ⁇ 1/ ⁇ complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2/ ⁇ complex and/or the GDP-bound form of RalA, and At least one reagent, such as a labeled antibody, binds to the biological product.
  • the kit can also include appropriate biological standards and control samples, to which the results of the experimental assay can be compared.
  • the present application also relates to a method for detecting the activity of the subject's RalGAP ⁇ -RalA signaling pathway, comprising: obtaining the body fluid or tissue of the subject; and detecting the ability of the first cell of the body fluid or tissue to transport Ca2+.
  • the above method further includes: obtaining the nucleic acid of a gene related to the RalGAP ⁇ -RalA signaling pathway; expressing the obtained nucleic acid in the second cell; and detecting the ability of the second cell to transport Ca2+.
  • said second cell is a Xenopus laevis oocyte.
  • the present application also relates to a method for diagnosing cardiomyopathy in a subject, comprising: providing a substance that interacts with proteins in the RalGAP ⁇ -RalA signaling pathway; cell, body fluid, or tissue exposure; and assessing the levels of proteins in the subject's RalGAP ⁇ -RalA signaling pathway.
  • the present application also relates to a method for diagnosing cardiomyopathy in a subject, comprising: detecting the ability of the first cell or the second cell to transport Ca 2+ by any of the methods described above.
  • FIGS 1A-I show the expression trend of RalGAP ⁇ 1 complex in pressure overloaded hearts according to one embodiment of the present invention
  • FIGS. 2A-H show that RalGAP ⁇ 1 deficiency according to one embodiment of the present invention causes cardiac dysfunction in mice and exacerbates TAC-induced cardiomyopathy;
  • FIGS. 3A-J show that SERCA2 is a target interacting with the RalGAP ⁇ 1 complex and that the RalGAP ⁇ 1 complex regulates SR Ca reuptake in cardiomyocytes according to one embodiment of the present invention
  • FIGS 4A-D show that the GDP-bound form of RalA regulates SERCA2 downstream of RalGAP ⁇ 1 according to one embodiment of the present invention
  • Figures 5A-L show that RalGAP ⁇ 1 and RalA-GDP promote oligomerization of SERCA2a according to one embodiment of the present invention
  • FIGS 6A-D show that expression of the GDP-binding form of the RalA S28N mutant protects against TAC-induced cardiomyopathy according to one embodiment of the present invention
  • Figure 7 shows a RalGAP ⁇ 1-RalA signaling pathway model according to one embodiment of the present invention.
  • Figure 8 shows that RalGAP ⁇ 2 deficiency according to one embodiment of the present invention results in impaired SR Ca 2+ reuptake in neonatal rat primary cardiomyocytes.
  • the term “about” refers to the variation in the value of a measurement, eg, volume, time, pressure, concentration, etc., due to the typical error rate of the equipment used to obtain the measurement. In one embodiment, the term “about” means within 5%-10% of the stated value; preferably, the term “about” means within 3%-5% of the stated value.
  • heart failure refers to a clinical syndrome characterized by typical symptoms such as dyspnea, ankle swelling, and fatigue.
  • heart failure may be accompanied by manifestations caused by structural and functional cardiac abnormalities (such as elevated jugular venous pressure, pulmonary crackles, and peripheral edema), resulting in reduced cardiac output and/or Increased pressure within the heart during stress.
  • AHF acute heart failure
  • CHF chronic heart failure
  • HFrEF reduced ejection fraction
  • HFmEF left ventricular ejection fraction
  • HFpEF preserved ejection fraction
  • HFmrEF and “HFpEF” include two additional criteria, elevated natriuretic peptide levels (BNP >35pg/ml and/or NT-proBNP >125pg/mL) with structural and/or functional heart disease Evidence (left ventricular hypertrophy and/or evidence of left atrial enlargement and/or diastolic dysfunction).
  • treatment refers to any indication of success in the treatment or amelioration of a disease or condition.
  • Treatment can include, for example, reducing or alleviating the severity of one or more symptoms of a disease or condition, or can include reducing the frequency of symptoms of a disease, defect, disorder, or adverse condition, etc., experienced by an individual, such as a human patient.
  • prevention refers to the prevention of a disease or disorder in an individual, such as a human patient. For example, if an individual at risk of developing heart failure does not develop heart failure after treatment with the methods of the invention, the disease has been prevented in that individual.
  • treat or prevent is sometimes used herein to refer to methods that result in some degree of treatment or amelioration of a disease or condition, and various outcomes directed to this end are contemplated, including but not limited to complete arrest of the condition.
  • active ingredient refers to a substance that provides a beneficial effect to an individual to which the substance is administered.
  • a “therapeutically effective amount” or “therapeutically effective dose” is an amount of a composition or active ingredient sufficient to provide a beneficial effect to the individual to which the composition or active ingredient is administered.
  • pharmaceutically acceptable carrier means a chemical composition which can be combined with an active ingredient or a mixture comprising an active ingredient and which, when combined, is useful for administering the compound to a mammal.
  • the term “carrier” will protect the biological product from rapid elimination from the body, as well as transport the biological product to a functional area.
  • the carrier can combine RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2/ ⁇ complex, RalA, RalA in GDP binding form, RalA S28N or RalA S28N in GDP binding form, SERCA2, SERCA2a oligomer and the above-mentioned Splice variants of proteins or nucleic acids, truncation, fragments, substitutions, addition or deletion mutants, recombination mutants, motif sequences, fusion proteins and homologues, homologous genes are delivered to the subject.
  • the carrier can have the characteristics of controlled and sustained release of the drug system, with ideal drug release speed and good sustained and controlled release effect.
  • the carrier may have good biocompatibility. Biodegradability and physiological properties.
  • first delivery carrier is mainly a polymer biological material, which is used to deliver biological products in the form of proteins or polypeptides, such as RalA containing RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2/ ⁇ complex, RalA, GDP binding form , RalA S28N or GDP binding form RalA S28N , SERCA2, SERCA2a oligomers and splice variants of the above proteins, truncation, fragment, substitution, addition or deletion mutants, recombination mutants, motif sequences, fusion proteins and homologues 1. Carrying the homologous gene into the subject.
  • the first delivery carrier includes: implants, liposomes, nanoparticles, hydrogels, microspheres, microencapsulated delivery systems, and nano biomedical carriers.
  • the term "second delivery vehicle” is mainly used for nucleic acid molecules, such as RalA comprising RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2/ ⁇ complex, RalA, GDP-bound form, RalA S28N or GDP-bound form RalA S28N , SERCA2 , SERCA2a, and the splice variants of the above nucleic acids, truncation, fragments, substitutions, addition or deletion mutants, recombinant mutants, motif sequences and homologues, DNA and RNA of homologous genes, etc., are carried into the subject, and Expressed in a subject or released into a subject after expression.
  • the second delivery carrier includes: adeno-associated virus (AAV), herpes virus, vaccinia virus, RNA virus such as retrovirus, colloidal dispersion system and nano biomedical carrier.
  • AAV adeno-associated virus
  • herpes virus herpes virus
  • a biological product of the invention may be included in a first delivery vehicle and/or a second delivery vehicle.
  • the first delivery vehicle is, for example, a controlled release formulation, including implants and liposomes, nanoparticles, hydrogels, microspheres, microencapsulated delivery systems, and the like.
  • the first delivery vehicle can use biodegradable, biocompatible polymers, such as polyesters, polyacrylates and their copolymers, such as polylactic acid and its copolymers, poly- ⁇ -hydroxycarboxylates, ethylene Vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, polyacrylic acid polymerized from monomers such as methacrylic acid, acrylate and acrylates such as butyl methacrylate and propylene glycol methacrylate wait.
  • biodegradable, biocompatible polymers such as polyesters, polyacrylates and their copolymers, such as polylactic acid and its copolymers, poly- ⁇ -hydroxycarboxylates, ethylene Vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester,
  • the first delivery vehicle is chitosan and other polysaccharides, alginate, collagen, gelatin, fibrin, glycosaminoglycan, agarose, and the like.
  • liposomal suspensions containing biologicals can also be used as pharmaceutically acceptable carriers.
  • the first delivery vehicle is a nanobiomedical vehicle.
  • the DNA encoding the biological product of the present invention can be introduced into cells, and then the biological product of the present invention encoded by the DNA can be expressed in the cell.
  • delivery of the biologicals of the invention can be achieved using a second delivery vehicle.
  • the second delivery vector is a recombinant expression vector such as a chimeric virus or a colloidal dispersion system or a nanobiomedical vector.
  • Various viral vectors that can be used to introduce DNA into cells include adenoviruses, herpes viruses, vaccinia viruses or RNA viruses such as retroviruses.
  • colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems.
  • Lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vesicles that can be used as delivery vehicles. RNA, DNA, or whole virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form. Methods for the efficient transfer of genes into cells using liposome vectors are known in the art.
  • the composition of liposomes generally includes a combination of phospholipids, typically in combination with a steroid, especially cholesterol, although other phospholipids or lipids may also be used.
  • RalGAP ⁇ -RalA signaling pathway refers to a signaling pathway in which RalGAP ⁇ protein interacts with the small G protein RalA to regulate the Ca 2+ transduction activity of cardiomyocyte SERCA2.
  • the RalGAP ⁇ protein may be RalGAP ⁇ 1 or RalGAP ⁇ 2.
  • Proteins of the RalGAP ⁇ -RalA signaling pathway include RalGAP ⁇ 1, RalGAP ⁇ 1/ ⁇ complex, RalGAP ⁇ 2, RalGAP ⁇ 2/ ⁇ complex, RalA, the GDP-bound form of RalA, and SERCA2.
  • RalGAP ⁇ 1-RalA signaling pathway refers to the signaling pathway in which RalGAP ⁇ 1 interacts with the small G protein RalA to regulate the Ca 2+ transduction activity of cardiomyocyte SERCA2.
  • GDP-bound RalA can directly regulate the Ca 2+ transduction activity of SERCA2, and the complex formed by the combination of RalGAP ⁇ 1 and RalGAP ⁇ can regulate the intrinsic activity of RalA, and then regulate the Ca 2+ transduction activity of SERCA2.
  • motif sequence refers to a conserved sequence in biological macromolecules such as DNA and protein, which is another structural level between the secondary and tertiary structures.
  • homologues refers to organic compounds that have similar structures but differ in molecular composition by several "CH2" atomic groups.
  • homeogene refers to a class of genes that contain a homeobox.
  • fusion protein has two different meanings, one is the expression product obtained by recombinant DNA technology after the recombination of two genes. Another meaning is a group of proteins that mediate the fusion of two cell plasma membranes,
  • expression cassette refers to a cassette containing a promoter that can specifically express Ral S28N in cardiomyocytes. In addition to the promoter and the Ral S28N gene, it also includes the carrier carrying the promoter and the gene.
  • complex refers to the association of two or more proteins. This association can be covalent or non-covalent, including, for example, ionic, hydrophilic and hydrophobic interactions between the two proteins in the complex. Typically, the proteins that form the complex interact with each other such that identification or detection of a first protein in the complex results in identification or detection of one or more other proteins that form the complex with the first protein. Protein complexes can be identified in vivo where two or more proteins naturally associate with each other to form a complex, eg, in a cell. Alternatively, a complex can be formed in vitro, wherein an interaction occurs between two or more proteins when these proteins are added to the same reaction mixture.
  • RalGAP ⁇ 1 genes with known sequences and partially known functions, or polypeptides expressed by genes ,protein.
  • the "RalGAP ⁇ 1" sequence can be found on the NCBI website Gene ID: 253959; the "RalGAP ⁇ 2" sequence can be found on the NCBI website Gene ID: 5718; the “RalGAP ⁇ ” sequence can be found on the NCBI website Gene ID: 57148; the “RalA” sequence can be found on the NCBI website Gene ID: 5898;
  • the "RalB” sequence please refer to the NCBI website Gene ID: 5899; for the "SERCA2” sequence, please refer to the NCBI website Gene ID: 488; for the "SERCA2a” sequence, please refer to the NCBI website Gene ID: 11938.
  • SERCA2 dysfunction is a hallmark of heart failure.
  • the present invention is based on the RalGAP ⁇ 1-RalA signaling model discovered by the inventors (refer to FIG. 7 ).
  • the RalGAP ⁇ 1 complex interacts with SERCA2 and positively regulates SERCA2 activity and SR Ca 2+ reuptake.
  • a downstream regulator of the RalGAP ⁇ 1 complex, the GDP-bound state of RalA determines the dynamic interaction with SERCA2.
  • This signaling model is critical for preserving cardiac function.
  • the present invention proposes to use the RalGAP ⁇ 1-RalA signaling model to regulate SERCA2 activity to prevent and treat cardiomyopathy or heart failure drugs.
  • such drugs can be biological products, including: proteins, protein complexes, polypeptides and antibodies.
  • the biologics of the present invention target the RalGAP ⁇ 1-RalA signaling pathway comprising SERCA2 in cardiomyocytes.
  • these drugs include RalGAP protein complexes, especially the complex RalGAP ⁇ 1/ ⁇ formed by the catalytic subunit RalGAP ⁇ 1 and the regulatory subunit RalGAP ⁇ , that is, the RalGAP ⁇ 1 complex, the complex formed by the catalytic subunit RalGAP ⁇ 2 and the regulatory subunit RalGAP ⁇ .
  • these agents include splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc. of RalGAP ⁇ 1, RalGAP ⁇ 2, RalGAP ⁇ 1 or RalGAP ⁇ 2 .
  • these agents include RalA, the GDP-bound form of RalA, and splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusions of RalA, the GDP-bound form of RalA Proteins, homologues, homologous genes, etc.
  • RalA S28N mutant for example: RalA S28N mutant.
  • the invention provides compositions comprising biological products of the invention. These compositions are suitable for pharmaceutical use and administration to patients.
  • the composition generally contains one or more biological products of the invention and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • the compositions of the biological products of the present invention may also contain other active compounds that provide complementary, additional or enhanced therapeutic functions.
  • the biologicals of the invention can be included in carriers that will protect the biologicals against rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are known to those skilled in the art.
  • liposomal suspensions containing biologicals can also be used as pharmaceutically acceptable carriers.
  • the DNA encoding the biological product of the present invention can be introduced into cells, and then the biological product of the present invention encoded by the DNA can be expressed in the cell.
  • delivery of the biologics of the invention can be achieved using recombinant expression vectors such as chimeric viruses or colloidal dispersion systems.
  • recombinant expression vectors such as chimeric viruses or colloidal dispersion systems.
  • viral vectors that can be used to introduce DNA into cells include adenoviruses, herpes viruses, vaccinia viruses or RNA viruses such as retroviruses.
  • colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems.
  • Lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vesicles that can be used as delivery vehicles. RNA, DNA, or whole virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form. Methods for the efficient transfer of genes into cells using liposome vectors are known in the art.
  • the composition of liposomes generally includes a combination of phospholipids, typically in combination with a steroid, especially cholesterol, although other phospholipids or lipids may also be used.
  • the biological product of the present invention is RalA S28N recombinant protein or a DNA molecule encoding RalA S28N recombinant protein, which is used to express or overexpress RalA S28N recombinant protein in cardiomyocytes.
  • the biological product of the present invention is an expression cassette for expressing the RalA S28N mutant driven by a Ctnt promoter in an adeno-associated virus (AAV) system.
  • the RalA S28N mutant was specifically expressed in the heart by intravenous injection of the AAV virus used to express the RalA S28N mutant.
  • the RalGAP ⁇ 1 complex/RalGAP ⁇ 2 complex plays a protective role in pressure overload-induced cardiomyopathy or heart failure.
  • RalGAP ⁇ 1 complex/RalGAP ⁇ 2 complex can positively regulate SERCA2 activity and accelerate SR Ca 2+ reuptake.
  • the GDP-bound form of RalA also has the same function as a downstream regulator of the RalGAP ⁇ 1 complex.
  • the cardiomyopathy or heart failure mentioned in this application is characterized by abnormal calcium ion transport mediated by SERCA2. In some embodiments, wherein the cardiomyopathy is heart failure. In some embodiments, wherein the cardiomyopathy is chronic heart failure.
  • these methods and uses comprise administering to a human subject an effective amount of a biological agent of the invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes and/or GDP-binding forms of RalA or its splice variants, truncation Short, fragment, substitution, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologs, homologous genes, etc.
  • a biological agent of the invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA or its splice variants, t
  • the above methods and uses also include encoding the biological product of the present invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP binding form RalA or its splice variant Body, truncation, fragment, substitution, addition and deletion mutation, recombination mutation, motif sequence, fusion protein, homologue, homologous gene and other DNA molecules are introduced into cardiomyocytes.
  • the biological product of the present invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP binding form RalA or its splice variant Body, truncation, fragment, substitution, addition and deletion mutation, recombination mutation, motif sequence, fusion protein, homologue, homologous gene and other DNA molecules are introduced into cardiomyocytes.
  • Phospholambans are well-known regulators of SERCA2, binding to and exerting an inhibitory effect on the Ca2 + pump.
  • RalA exerts a positive regulation on SERCA2 after binding to the Ca2 + pump. Both phospholamban and RalA are tightly regulated and exhibit dynamic interactions with SERCA2.
  • the present invention also proposes a method and application for regulating the intracellular balance of Ca 2+ in cardiomyocytes by regulating the function of SERCA2.
  • the intracellular balance of Ca 2+ can be dynamically adjusted positively or negatively through the interaction with SERCA2.
  • the above methods and uses comprise administering to a human subject an effective amount of a biological agent of the present invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or RalA and/or or GDP-bound RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc.
  • a biological agent of the present invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or RalA and/or or GDP-bound RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc.
  • the above methods and uses also include encoding the biological product of the present invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or RalA and/or GDP binding form RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc. in cardiomyocytes.
  • the biological product of the present invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or RalA and/or GDP binding form RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc. in cardiomyocytes.
  • RalA The GDP-bound form of RalA causes oligomerization of SERCA2, increasing the Ca2 + transport activity of SERCA2 but not its ATPase activity. However, this interaction with the GDP-bound form of RalA alters SERCA2 configuration to promote oligomerization of the Ca2 + pump independent of Thr 484 phosphorylation.
  • the present invention also relates to methods and uses for up-regulating oligomerization of SERCA2a.
  • SERCA2a oligomerization is positively regulated by administering to cardiomyocytes an effective amount of the GDP-bound form of RalA.
  • these methods and uses include administering to a human subject an effective amount of a compound of formula I or a composition containing a compound of formula I; or administering to a human subject an effective amount of a biological agent of the present invention, such as RalGAP ⁇ 1 and/or or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form of RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, Homologs, homologous genes, etc.
  • the above methods and uses also include encoding the biological product of the present invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or GDP binding form RalA or its splice variant, truncation, fragment, substitution, addition DNA molecules such as deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc., such as RalA S28N or GDP-binding form RalA S28N , are introduced into cardiomyocytes.
  • the biological product of the present invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or GDP binding form RalA or its splice variant, truncation, fragment, substitution, addition DNA molecules such as deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc., such as RalA S28N or GDP-binding form RalA S28N , are introduced into cardiomyocytes.
  • the RalGAP ⁇ -RalA signaling channel model plays a role by regulating SERCA2, which is essential for Ca 2+ intracellular balance and maintaining heart function. It not only plays a protective role in heart failure caused by pressure overload, but also plays a role in the treatment of secondary heart failure. It also has preventive and therapeutic significance. Therefore, the present invention also proposes the method and application of using RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 complex and/or GDP-bound RalA to prevent and treat cardiomyopathy or heart failure.
  • these methods and uses comprise administering to a human subject an effective amount of a biological agent of the invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc.
  • a biological agent of the invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA or its splice variants, truncations, fragments, substitutions, addition and deletion mutations, recombination mutations, motif sequences, fusion proteins, homologues, homologous genes, etc.
  • the above methods and uses also include encoding the biological product of the present invention, such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP binding form RalA or its splice variant Body, truncation, fragment, substitution, addition and deletion mutation, recombination mutation, motif sequence, fusion protein, homologue, homologous gene and other DNA molecules are introduced into cardiomyocytes.
  • the biological product of the present invention such as RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP binding form RalA or its splice variant Body, truncation, fragment, substitution, addition and deletion mutation, recombination mutation, motif sequence, fusion protein, homologue, homologous gene and other DNA molecules are introduced into cardiomyocytes.
  • the invention provides methods and uses of upregulating SERCA2 activity in cardiomyocytes; in particular, methods and uses of upregulating SR Ca2 + reuptake by SERCA2 in cardiomyocytes. Further, in some embodiments, the present invention also provides regulating the intracellular balance of Ca 2+ in cardiomyocytes by regulating the function of SERCA2. In some embodiments, the present invention also provides methods and uses for up-regulating oligomerization of SERCA2a. In some embodiments, the present invention provides methods and uses for the prevention and treatment of cardiomyopathy or heart failure.
  • the method for positively regulating SERCA2a oligomerization of the present application includes: administering to a subject an effective amount of any one of the above pharmaceutical compositions.
  • the method for positively regulating SERCA2 activity of the present application includes: administering an effective amount of any one of the above pharmaceutical compositions to the subject.
  • the method of the present application for preventing and treating cardiomyopathy in a subject includes: administering an effective amount of any one of the above pharmaceutical compositions to the subject.
  • the cardiomyopathy is caused by abnormal calcium ion transport mediated by SERCA2. Wherein the cardiomyopathy is heart failure, further, the cardiomyopathy is chronic heart failure.
  • the present invention also provides a diagnostic tool that can be used to quantitatively or qualitatively detect SERCA2 activity.
  • a biological product such as an antigen, that interacts with RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or RalAGDP and/or bound form RalA is provided.
  • These biologicals can be fluorescently labeled.
  • Samples can be incubated with labeled biologicals, excess unbound protein can be washed away, and the tissue can then be assessed for fluorescent activity, which will indicate the presence of RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complexes and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complexes Object and/or GDP binding form RalA.
  • biological products that interact with RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complexes and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complexes and/or GDP-bound RalA can be used to detect the presence of RalGAP ⁇ 1 complexes in cells, body fluids, tissues or organisms compound and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA and its content.
  • the detected amount of RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA can be correlated with the activity of SERCA2.
  • the present invention also includes a kit for detecting the level of RalGAP ⁇ 1 and/or RalGAP ⁇ 1 complex and/or RalGAP ⁇ 2 and/or RalGAP ⁇ 2 complex and/or GDP-bound form RalA in a sample, which comprises at least one compound with RalGAP ⁇ 1 and/or RalGAP ⁇ 1 a complex and/or RalGAP ⁇ 2 and/or a RalGAP ⁇ 2 complex and/or a GDP-bound form RalA interacting biological product, such as an antigen, whether it is labeled or unlabeled, and at least one reagent that binds to the biological product, For example labeled antibodies.
  • the kit can also include appropriate biological standards and control samples, to which the results of the experimental assay can be compared.
  • the method for quantitatively or qualitatively detecting SERCA2 activity in a subject includes: providing a substance that interacts with proteins in the RalGAP ⁇ -RalA signaling pathway, the substance can be a gene, protein, polypeptide, or an organic compound, compound composition etc. Contacting the cells, body fluids or tissues of the subject with the substance triggers the interaction between the substance and any protein, polypeptide or gene in the RalGAP ⁇ -RalA signaling pathway. Evaluate the protein content in the RalGAP ⁇ -RalA signaling pathway of the subject.
  • the protein to be evaluated can be any protein in the RalGAP ⁇ -RalA signaling pathway.
  • the method for detecting the activity of the RalGAP ⁇ -RalA signaling pathway in a subject comprises: obtaining body fluid or tissue of the subject, and the body fluid or tissue includes all components of the RalGAP ⁇ -RalA signaling pathway. If the subject's heart muscle is healthy, the obtained body fluid or tissue cells have normal calcium ion transport activity; or the subject has myocardial disease, such as heart failure, the obtained body fluid or tissue cells cannot normally transport calcium ion. The ability to transport Ca 2+ in first cells of body fluids or tissues is tested. Wherein, the first cell is a cell obtained from the body fluid or tissue of the subject.
  • detecting the activity of the RalGAP ⁇ -RalA signaling pathway in the subject further includes acquiring the nucleic acid of a gene related to the RalGAP ⁇ -RalA signaling pathway; and expressing the acquired nucleic acid in the second cell.
  • the second cell is a living cell capable of expressing genes in vitro, such as Xenopus laevis oocyte. The ability of the second cell to transport Ca2 + was tested.
  • the ability of the first cell or the second cell to transport calcium ions can be detected using electrophysiological techniques. In some embodiments, the ability of the first cell or the second cell to transport calcium ions can be detected using patch clamp techniques.
  • the method for diagnosing cardiomyopathy in a subject comprises: providing a substance that interacts with a protein in the RalGAP ⁇ -RalA signaling pathway; contacting cells, body fluids, or tissues of the subject with the substance; evaluating the subject for RalGAP ⁇ - Content of proteins in the RalA signaling pathway.
  • whether the subject suffers from cardiomyopathy can be diagnosed by detecting and evaluating the ability of the RalGAP ⁇ -RalA signaling pathway to transport Ca 2+ in the first cell or the second cell.
  • Example 1 Increased expression of the RalGAP ⁇ 1 complex in pressure overloaded hearts
  • mice used in this and subsequent examples were fed with a light/dark cycle of 12 hours under specified pathogen-free conditions. Unless otherwise stated, mice had free access to food and water in their cages. Pressure overloaded hearts were constructed by performing TAC surgery on anesthetized male mice (2-3 months old). Specifically, ketamine (100 mg/kg) and xylazine (10 mg/kg) were injected intraperitoneally into mice.
  • the arch aorta was surgically exposed and ligated with 6-0 suture using a 27-gauge needle to create a constriction with a diameter of 0.413 mm.
  • mice were sacrificed to isolate heart tissue. Before lysis, mouse heart tissue was snap-frozen in liquid nitrogen and stored at minus 80 °C. Tissue lysates were then obtained by homogenization in lysis buffer and lysis on ice for 30 minutes to remove tissue debris by centrifugation.
  • Immunoprecipitation of the target protein was performed by using antibody-coupled protein G-Sepharose or GFP-binding agent at 4°C for 16 hours. The resin was washed to remove non-specifically bound proteins, and the immunoprecipitated complexes were eluted in SDS sample buffer.
  • Lysates or immunoprecipitates are separated by electrophoresis. After electrophoresis, separated proteins were immunoblotted onto nitrocellulose membranes and incubated with primary antibodies. After further probing with a horseradish peroxidase (HRP)-conjugated secondary antibody, the membrane was incubated with HRP substrate to enhance chemiluminescence.
  • HRP horseradish peroxidase
  • Immunoprecipitated complexes were separated by SDS-PAGE and visualized by Coomassie blue staining. Then, the protein bands of interest were excised on the gel and subjected to in-gel digestion using trypsin as the digestive enzyme, followed by analysis of each gel band by LC-MS/MS.
  • Reagents were used to extract total RNA and use RT kit for reverse transcription into cDNA. Perform QPCR analysis of the gene of interest using the Roche Lightcycler Real-Time PCR System and the primers listed in Table 2. Data analysis for multiple groups was performed by t-test for two groups or by one-way or two-way ANOVA using Prism software. Differences were considered statistically significant at p ⁇ 0.05.
  • ⁇ MHC was increased at the protein level in pressure overloaded hearts (see FIG. 1A ).
  • Ralgap ⁇ 1 and Ralgap ⁇ mRNA levels were comparable in sham and TAC-operated hearts whereas protein levels of both were significantly increased in TAC-operated hearts (see Figures 1A-C).
  • both mRNA and protein levels of RalGAP ⁇ 2 were unchanged in TAC-operated hearts (see Figure 1A-C).
  • RalA and RalB downstream of the RalGAP complex remained normal at both mRNA and protein levels (see Figure 1A-C).
  • Angiotensin II (Ang-II) and norepinephrine (NE) are two key regulators of neurohormones in pressure overload-induced cardiomyopathy.
  • NE treatment increased RalGAP ⁇ 1 protein, but not its mRNA level, in primary neonatal rat ventricular cardiomyocytes (NRVC) (see Figure 1D-F).
  • NRVC neonatal rat ventricular cardiomyocytes
  • Ang-II stimulation increased its protein levels in primary NRVC (see Figure 1G-I).
  • Example 2 RalGAP ⁇ 1 deficiency causes cardiac dysfunction and exacerbates TAC-induced cardiomyopathy
  • Cardiomyocytes and fibroblasts are two cell populations in the heart. Although both are present in the heart, both RalGAP ⁇ 1 and ⁇ 2 are preferentially expressed in cardiomyocytes, but not in cardiac fibroblasts (see Figure 2A).
  • the inventors generated a mouse model of RalGAP ⁇ 1 cardiomyocyte-specific deletion (RalGAP ⁇ 1-cKO).
  • RalGAP ⁇ 1 f/f mouse obtained by reference: Chen Q, Rong P, Zhu S, Yang X, Ouyang Q, Wang HY, Chen S. Targeting ralgapalpha1 in skeletal muscle to simultaneously improve postprandial glucose and lipid control. Sci Adv. 2019; 5: eaav4116
  • ⁇ MHC-Cre mice for which references can be obtained: Agah R, Frenkel PA, French BA, Michael LH, Overbeek PA, Schneider MD. Gene recombination in postmitotic cells. Targeted expression of cre recombinase provokes cardiac -restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest.
  • RalGAP ⁇ 1-cKO mice cardiomyocyte-specific RalGAP ⁇ 1 knockout mice
  • RalGAP ⁇ 1 f/f X RalGAP ⁇ 1 f/f -Cre matings were set up to generate RalGAP ⁇ 1 f/f (control mice) and RalGAP ⁇ 1 f/f -Cre (RalGAP ⁇ 1-cKO mice).
  • RalGAP ⁇ 1 f/f mice were genotyped using the following primers:
  • Cre mice were genotyped using the following primers:
  • RalGAP ⁇ 1 f/f was reduced in the heart but not altered in other tissues, including skeletal muscle, liver, brown adipose tissue, and white adipose tissue (see Fig. 2B).
  • Specific deletion of RalGAP ⁇ 1 in cardiomyocytes did not affect the expression of RalGAP ⁇ 2 (see Figure 2B). Since the stability of RalGAP ⁇ depends on RalGAP ⁇ , RalGAP ⁇ showed a modest reduction in the heart but remained normal in other tissues (see Figure 2B).
  • Example 3 SERCA2 is a target interacting with the RalGAP ⁇ 1 complex and the RalGAP ⁇ 1 complex regulates SR Ca 2+ reuptake in cardiomyocytes
  • the proteomics method was used to identify the interacting proteins of the RalGAP ⁇ 1 complex, so as to clarify the mechanism of the RalGAP ⁇ 1 complex regulating cardiac function.
  • GFP-RalGAP ⁇ 1 and HA-RalGAP ⁇ were expressed in HEK293 cells, which were subsequently immunoprecipitated using GFP-Trap beads. Proteins co-immunoprecipitated with GFP-RalGAP ⁇ 1 were identified by mass spectrometry.
  • Human embryonic kidney HEK293 cells were purchased from the Chinese Academy of Medical Sciences and Peking Union Medical College Cell Resource Center (China), maintained in DMEM medium containing 10% (v/v) fetal bovine serum, and regularly tested for mycoplasma. Cell transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific).
  • Primary neonatal rat cardiomyocytes were obtained from the ventricles of neonatal Sprague Dawley rats (postnatal days 0-3). Ventricles were isolated and cubed from neonatal rats. Sequential digestion of ventricular squares with trypsin. Filter the cell suspension through a cell strainer (70 ⁇ m mesh size) to remove tissue debris. The resulting cells were seeded in DMEM containing 10% (v/v) fetal calf serum for 1 hour to allow the fibroblasts to settle for removal. Cardiomyocytes were then plated in fresh DMEM supplemented with 10% (v/v) fetal bovine serum. Primary neonatal cardiomyocytes were transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific).
  • a Fluo-4-AM-based method measures calcium transients in cardiomyocytes.
  • Cardiomyocytes were cultured in Hanks buffer containing 1 mM. Load Fluo-4-AM and use GRASS S48 stimulator to electrically stimulate cardiomyocytes.
  • Line scan images were taken using a Zeiss LSM880 confocal microscope and analyzed with IDL5.5 (Harris Geospatial Solutions); where the decay time was defined as the time from peak to 63% of the peak to basal level of the decay phase of the calcium transient.
  • HEK293 cells expressing SERCA2a were loaded with 5 ⁇ M Fluo-4-AM and stimulated with 100 ⁇ M ATP.
  • Frame scan images of cells were taken using Olympus.
  • T-tubule (TT) organization in cardiomyocytes was analyzed using a Di-8-ANEPPS-based imaging method. Isolated primary cardiomyocytes were stained with Di-8-ANEPPS.
  • Cell images were obtained using a Carl Zeiss880 confocal microscope, and fast Fourier transform was performed on the obtained images.
  • TT power peak amplitude
  • microsomes containing crude SR membrane vesicles were isolated for detection of SERCA2-ATPase activity in microsomes.
  • ATP hydrolysis reactions were performed by incubating microsomes (50 ⁇ g protein) in assay buffer containing 100 mM KCl, 10 mM HEPES (pH 7.4), 5 mM MgCl2, 100 ⁇ M CaCl2, 1.5 mM ATP, 2 ⁇ M A23187, and 5 mM sodium azide. Hold at 30°C for 30 min and terminate by addition of ice-cold 10% TCA.
  • Hydrolysis reactions were performed in the absence or presence of 5 [mu]M thapsigargin to determine total and thapsigargin-insensitive calcium pump quantities, respectively.
  • the thapsigargin-sensitive active Ca 2+ -ATPase (SERCA2-ATPase) was determined by subtracting the thapsigargin-insensitive Ca 2+ -ATPase activity from the total activity.
  • microsomes containing crude SR membrane vesicles were isolated to examine Ca2 + uptake in microsomes.
  • Ca2 + uptake was measured using a Fura-2-based method.
  • Microsomes were resuspended in assay buffer (100 mM KCl, 10 mM HEPES-KOH (pH 7.4), 10 mM oxalate, 5 mM MgCl2 and 10 ⁇ M ruthenium red) containing 2 ⁇ M Fura-2 free acid.
  • ATP 5 mM
  • Ca 2+ (2 ⁇ M
  • Double excitation was performed at 340 nM and 380 nM and the emitted fluorescence was recorded at 510 nM using a fluorescent microplate reader.
  • Free Ca 2+ was plotted using Clampfit 10.4 (Molecular Devices), and Ca 2+ uptake rates were calculated using the linear portion of the curve after onset of absorption.
  • RalGAP ⁇ a known binder of RalGAP ⁇ 1
  • SERCA2 a key regulator of cardiac Ca homeostasis
  • FIG. 3C endogenous SERCA2 co-immunoprecipitated with endogenous RalGAP ⁇ 1 immunoprecipitated from cardiac lysates
  • RalGAP ⁇ 1 was overexpressed in HEK293 cells, and Ca transients induced by the addition of ATP were measured.
  • the full duration at half maximum (FDHM) and time constant Tau of Ca transients are two measures that reflect the rate of Ca reuptake into the ER, and the peak is a measure of Ca in the cytosol.
  • FDHM full duration at half maximum
  • Tau time constant Tau of Ca transients
  • Overexpression of RalGAP ⁇ 1 resulted in a significant decrease in FDHM and Tau of Ca transients in HEK293 cells, indicating accelerated Ca reuptake into the ER (see Figure 3D).
  • the peak value of Ca 2+ transients was significantly increased, indicating a decrease in cytosolic Ca 2+ (see Figure 3D).
  • RalGAP ⁇ 1 in NRVC was knocked down using small interfering RNA (siRNA), and the effect of RalGAP ⁇ 1 reduction on Ca transients was examined. It was found that FDHM and Tau of Ca transients were significantly increased, whereas their peaks were suppressed in RalGAP ⁇ 1-reduced NRVC (see Figure 3E). Thus, the inventors found that the interaction of the RalGAP ⁇ 1 complex with SERCA2 suggests that it regulates Ca 2+ homeostasis by controlling SR Ca 2+ reuptake.
  • Cardiomyocyte-specific deficiency of RalGAP ⁇ 1 did not affect the mRNA levels of Ltcc, Ncx, and Ryr2 in the heart.
  • the expression of SERCA2a was also normal at both mRNA and protein levels.
  • Primary cardiomyocytes were isolated and Ca2 + homeostasis in these cells was analyzed.
  • T-tubules are invaginated sarcolemma in cardiomyocytes that contain membrane microdomains rich in ion channels and transporters. TT formed a branched and interconnected network whose regularity was roughly comparable between RalGAP ⁇ 1-deficient and control cells.
  • RalGAP ⁇ 1 regulates Ca homeostasis in cardiomyocytes by regulating SERCA2 - mediated SR Ca reuptake. Deficiency in RalGAP ⁇ 1 prolongs SR Ca reuptake in cardiomyocytes, which underlies cardiac dysfunction in RalGAP ⁇ 1-cKO mice.
  • Example 4 The GDP-bound form of RalA regulates SERCA2 downstream of RalGAP ⁇ 1
  • RalGAP ⁇ 1 includes a functional GAP domain with Asn 1949 as a key residue for activity.
  • Asn 1949 Lys mutation was introduced to inactivate its GAP activity, RalGAP ⁇ 1 lost its ability to accelerate Ca reuptake into the ER in a Ca transient assay.
  • the Asn 1949 Lys mutant of RalGAP ⁇ 1 neither decreased FDHM and Tau nor increased the magnitude of Ca2 + transients compared with wild-type RalGAP ⁇ 1 (see Figure 3D).
  • RalGAP ⁇ 1 converts these two small G proteins from the GTP-bound form to the GDP-bound form.
  • RalA but not RalB, interacts with SERCA2 when co-expressed in HEK293 cells (see Figure 4A).
  • the GDP-binding RalA S28N mutant but not the GTP-binding RalA G23V mutant was able to interact with SERCA2 when co-expressed in cells (see Figure 4A).
  • Expression of RalA was approximately comparable between hearts of RalGAP ⁇ 1-cKO and RalGAP ⁇ 1 f/f mice, while the GTP-bound form of RalA increased as expected in RalGAP ⁇ 1-deficient hearts (see Figure 4B).
  • the GDP-bound form of RalA may be reduced in RalGAP ⁇ 1-deficient hearts. More importantly, expression of the RalA S28N mutant, but not the RalA G23V mutant, decreased FDHM and Tau and increased Ca in HEK293 cells ( Figure 4C) or primary neonatal rat cardiomyocytes ( Figure 4D). 2+ magnitude of the transient. This suggests that the GDP form of RalA is capable of accelerating Ca reuptake into the SR. These data suggest that RalGAP ⁇ 1 regulates SERCA2a through the GDP-bound form of RalA.
  • oligomerization of SERCA2a was further investigated.
  • SERCA2a switches between monomeric and oligomeric, and oligomerization enhances its Ca2 + -transporting activity.
  • visual blots of SDS gels showed that the oligomerization of SERCA2a was significantly increased when it was co-expressed with the RalGAP ⁇ 1/ ⁇ complex in HEK293 cells (see Figure 5A).
  • SERCA2a oligomerization was significantly reduced in RalGAP ⁇ 1-cKO hearts (see FIG. 5B ). The reduction in oligomerization of SERCA2a is not due to Thr484 phosphorylation.
  • this example studies whether RalA has a direct effect on SERCA2a to regulate its Ca 2+ transport activity.
  • GST-RalA S28N recombinant protein was expressed and purified from E. coli and found to interact with Flag-SERCA2a in a GST-fold assay (see FIG. 5K ).
  • this GST-RalA S28N recombinant protein was added to microsomes purified from Flag-SERCA2a-expressing HEK293 cells, the GST-RalA S28N recombinant protein increased the Ca transport activity of SERCA2a (see Fig. 5L). This suggests that RalA directly activates Ca transport by SERCA2a .
  • RalGAP ⁇ 1 promotes SERCA2 oligomerization through GDP-bound RalA to enhance the transport activity of the Ca2 + pump.
  • RalGAP ⁇ 1 can also regulate the ATPase activity of SERCA2a through direct interaction with Ca2 + pump.
  • an expression cassette driven by a Ctnt promoter in an adeno-associated virus (AAV) system was constructed to specifically express the RalA S28N mutant in the heart.
  • AAV adeno-associated virus
  • AAV infection in the control group did not improve cardiac dysfunction induced by TAC surgery.
  • Both EF and FS of the AAV-infected control group were significantly lower in the TAC group compared with the sham group at 6 weeks after surgery (see FIG. 6D ).
  • TAC-induced decline in EF and FS was prevented in the TAC group infected with AAV expressing the RalA S28N mutant (see Figure 6D). This suggests that expression of RalA S28N contributes to the maintenance of cardiac function in the hearts of pressure-overloaded mice.
  • Example 7 RalGAP ⁇ 2 Deficiency Causes Impaired SR Ca2+ Reuptake in Neonatal Rat Primary Cardiomyocytes
  • SERCA2 was found in immunoprecipitates of RalGAP ⁇ 2 (see Figure 8A).
  • the inventors knocked down RalGAP ⁇ 2 in NRVC using small interfering RNA (siRNA) and examined the effect of RalGAP ⁇ 2 reduction on Ca2 + transients. It was found that FDHM and Tau of Ca transients were significantly increased, whereas their peaks were suppressed in RalGAP ⁇ 2-reduced NRVC (see Figure 8B). The increase in Ca 2+ transient FDHM and Tau caused by the reduction of RalGAP ⁇ 2 was reversed by RalA S28N (see Fig. 8C).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cardiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供一种药物，所述药物能够靶向心肌细胞中SERCA2，正向调节SERCA2的活性；特别地能够正向调节心肌细胞中SERCA2的SR Ca 2+再摄入。所述药物能够预防和治疗人受试者或其他脊柱动物的心肌病或心脏衰竭。

Description

人类受试者心肌病的治疗 技术领域
本发明涉及一种人类受试者的心肌病的治疗，特别地涉及一种利用RalGAPα-RalA信号模型正向调节心肌细胞中SERCA2的活性，从而对心脏提供保护。
背景技术
高血压及其相关的心血管和心脏疾病是世界范围内最主要的导致死亡的原因之一。高血压引起的压力超负荷能引起心脏功能障碍并导致心脏衰竭(Heart Failing)。发生心脏衰竭的计算终生风险预期随年龄增加将会增加。从60岁以下人群的低于2％到75岁以上人群的超过10％(Metra M,Teerlink JR,Lancet 2017；390:1981-1995)。而且，患有高血压的人处于心脏衰竭的较高风险中(Lloyd-Jones DM等，Circulation 2002；106:3068–3072)。心脏衰竭的患者具有不良预后，住院率和死亡率都较高。然而，高血压引起心脏衰竭的病理机制尚不清楚。
钙离子(Ca 2+)在肌浆网(Sarcoplasmic Reticulum,SR)和心肌细胞胞液之间的循环决定了心脏收缩活动。压力超负荷影响心脏中Ca 2+的循环并导致胞液中Ca 2+增加，减弱了心脏的伸缩力。因此，无论心脏衰竭的触发机制是什么，细胞内SR的Ca 2+摄取减少所引起的细胞内Ca 2+的异常分布都是一个基础因素(Schwinger RH等，J Mol Cell Cardiol.1999；31(3):479-91；Bers D等，Ann N.Y.Acad Sci 2006；1080:165-177)。
肌浆网/内质网Ca 2+ATP酶2(SERCA2)是一种ATP依赖的Ca 2+转运泵，是调节从胞液中Ca 2+的再摄取关键的酶。SERCA2的功能障碍在心脏衰竭中非 常明显。已知SERCA2的功能受到多种机制的正向和负向调控。这些调控机制包括磷蛋白结合、磷酸化和类泛素化修饰。例如，磷蛋白的结合能够对SERCA2的活性产生抑制作用，减慢SR中Ca 2+的再摄取。作为对照的，通过横纹肌优先表达的蛋白激酶(SPEG)的SERCA2Thr 484磷酸化则能增强Ca 2+转运活性。类似地，SERCA2的类泛素化修饰能够维持ATPase的活性和稳定性，而其在衰竭的心脏中则明显减少。
由RalA和RalB组成的Ral-GTP酶是多种细胞过程的关键调控因子。虽然RalA和RalB有些重复，但是二者在许多过程中具有不同的功能。RalA和RalB的内在活性由其上游的调控因子Ral-GTP酶激活蛋白(RalGAP)复合物调控。RalGAP复合物将这两个小G蛋白RalA和RalB从GTP结合状态转换到GDP结合形式。已知两种催化作用的RalGAPα1和RalGAPα2能够分别与调控作用的RalGAPβ结合形成RalGAP复合物-1(或者RalGAPα1复合物，二者具有相同含义)和RalGAP复合物-2(或者RalGAPα2复合物，二者具有相同含义)。虽然RalGAP复合物-1和RalGAP复合物-2都在心脏中表达，但它们在心脏中的功能仍然是未知的。
发明内容
本发明涉及预防和治疗心肌病或心脏衰竭药物。在一些实施例中，本发明的药物能够靶向心肌细胞中SERCA2，正向调节SERCA2的活性；特别地能够正向调节心肌细胞中SERCA2的SR Ca 2+再摄入。在一些实施例中，本发明的药物本身或者与其他药物联合通过调节SERCA2的功能调控心肌细胞中Ca 2+的胞内平衡。在一些实施例中，本发明的药物能够正向调节SERCA2a寡聚化。在一些实施例中，本发明的药物能够预防和治疗人受试者或其他脊柱动物的心肌病或心脏衰竭。在一些实施例中，本发明的药物可以是生物制品，包括：蛋白、蛋白复合物、多肽和抗体。
在本发明的一个方面，本申请涉及一种药物组合物在制备预防或治疗心肌病方面药物的应用，其中所述药物组合物可以包括：RalGAPα1、RalGAPα2、RalGAPα1/β复合物、RalGAPα2/β复合物、RalA、GDP结合形式的RalA，或者它们的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
特别地，所述心肌病由SERCA2介导的钙离子转运异常表征，例如心脏衰竭，特别是慢性心脏衰竭。
在本发明的另一个方面，本申请涉及一种药物组合物在制备正向调节SERCA2a寡聚化药物的应用，其中所述药物组合物包括：RalGAPα1、RalGAPα2、RalGAPα1/β复合物、RalGAPα2/β复合物、RalA、GDP结合形式的RalA，或者它们的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
在本发明的另一个方面，本申请涉及一种药物组合物在制备正向调节SERCA2活性药物的应用，其中所述药物组合物包括：其中所述药物组合物包括：RalGAPα1、、RalGAPα2、RalGAPα1/β复合物、RalGAPα2/β复合物、RalA、GDP结合形式的RalA，或者它们的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
本发明还提供了包含本发明生物制品或编码本发明生物制品的核酸分子组合物。这些组合物可适用于药物用途和对患者的给药。该组合物通常含有一种或多种本发明生物制品和药学上可接受的赋形剂。本文所用短语“药学上可接受的赋形剂”包括与药物给药相容的溶剂、分散介质、包衣、抗菌剂和抗真菌剂、等渗剂和吸收延迟剂等。这种介质和试剂用于药物活性物质的用途在本领域中是已知的。本发明生物制品的组合物还可包含提供补充，附加或增强治疗功能的其它活性化合物。
在一些实施例中，本发明的生物制品或者编码本发明生物制品的核酸分子可以包括在载体中，所述载体将保护生物制品或编码这些生物制品的核酸分子免于从体内迅速消除，和/或将生物制品递送至指定组织、器官。
在本发明的一个方面，本申请涉及一种药物组合物，包括：第一递送载体以及以下活性物质中的一种或多种：(1)RalGAPα1；(2)RalGAPα1/β复合物；(3)RalGAPα2；(4)RalGAPα2/β复合物；(5)RalA、GDP结合形式的RalA、或者(1)-(5)的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
特别地，第一递送载体可以为例如控释制剂，包括植入物和脂质体、纳米粒、水凝胶、微球、微囊化递送系统等。第一递送载体可以使用可生物降解的，生物相容的聚合物，例如聚酯类、聚丙烯酸酯类及其共聚物，如聚乳酸及其共聚物、聚-β-羟基羧酸酯、乙烯乙酸乙烯酯、聚酐、聚乙醇酸、胶原蛋白、聚原酸酯、由甲基丙烯酸、丙烯酸盐及丙烯酸酯如甲基丙烯酸丁酯、甲基丙烯酸丙二醇酯等单体聚合而成的聚丙烯酸等。制备这种制剂的方法对于本领域技术人员来说是已知的。在一些实施例中，第一递送载体为壳聚糖及其他多糖、藻酸盐、胶原、明胶、纤维蛋白、糖胺多糖、琼脂糖等。在一些实施例中，含有生物制品的脂质体悬浮液也可用作药学上可接受的载体。在一些实施例中，第一递送载体为纳米生物医学载体。
在本发明的另一个方面，本申请涉及一种药物组合物，包括：第二递送载体以及编码并能够在心肌细胞中表达以下活性物质中的一种或多种的核酸分子：(1)RalGAPα1；(2)RalGAPα1/β复合物；(3)RalGAPα2；(4)RalGAPα2/β复合物；(5)RalA、GDP结合形式的RalA；或者(1)-(5)的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
如上所述的药物组合物可将编码本发明生物制品的DNA导入细胞，然后 在细胞中表达由核酸分子编码的本发明的生物制品。特别地，本发明的生物制品的递送可使用第二递送载体实现。第二递送载体为重组表达载体如嵌合病毒或胶体分散系统或纳米生物医学载体、可用于将DNA导入细胞的各种病毒载体包括腺病毒、疱疹病毒、牛痘病毒或RNA病毒如逆转录病毒。在一些实施例中，胶体分散体系包括高分子复合物、纳米胶囊、微球、珠粒和脂质基体系。脂质基体系包括水包油乳液、胶束、混合胶束和脂质体。脂质体是可用作递送载体的人工膜囊泡。RNA、DNA或者完整的病毒粒子可以包封在含水的内部，并以生物活性形式递送到细胞。使用脂质体载体将基因有效转移到细胞中的方法是本领域已知的。脂质体的组合物通常包括磷脂的组合(典型地与类固醇，特别是胆固醇组合)，也可以使用其它磷脂或脂质。
特别地，在某些实施例中，本发明的生物制品为RalA S28N重组蛋白或者编码RalA S28N重组蛋白的核酸分子，其用于在心肌细胞中表达或过表达RalA S28N重组蛋白。
特别地，在某些实施例中，本发明的生物制品为腺相关病毒(AAV)系统中使用Ctnt启动子驱动的用于表达RalA S28N突变体的表达盒。通过静脉注射用于表达RalA S28N突变体的AAV病毒，能够在心脏中特异性表达RalA S28N突变体。
在一些实施例中，本发明提供正向调节心肌细胞中SERCA2活性的方法和用途；特别地，正向调节心肌细胞中SERCA2的SR Ca 2+再摄入的方法和用途。进一步地，在一些实施例中，本发明还提供通过调节SERCA2的功能调控心肌细胞中Ca 2+的胞内平衡。在一些实施例中，本发明还提供了正向调节SERCA2a寡聚化的方法和用途。在一些实施例中，本发明提供利用预防和治疗心肌病或心脏衰竭的方法和用途。
在一些实施例中，本申请涉及一种正向调节SERCA2a寡聚化的方法，包括：给予受试者有效量的如上任一所述的药物组合物。在一些实施例中，本申 请涉及一种正向调节SERCA2活性的方法，包括：给予受试者有效量的如上任一所述的药物组合物。在一些实施例中，本申请涉及一种预防和治疗受试者心肌病的方法，包括：给予受试者有效量的如上任一所述的药物组合物。
本申请还涉及一种鉴定能够影响RalGAPα-RalA信号通路的物质的方法，包括：提供RalGAPα-RalA信号通路中蛋白质的多肽或其片段、衍生物、同源物或突变体；在允许5’-ATP、5’-GTP和/或5’-GDP与所述多肽结合的条件下，将所述多肽或其片段、衍生物、同源物或突变体与测试物质接触；以及确定所述测试物质是否与所述多肽或其片段、衍生物、同源物或突变体结合。其中所述RalGAPα-RalA信号通路中蛋白质包括RalGAPα1、RalGAPα1/β复合物、RalGAPα2、RalGAPα2/β复合物、RalA、GDP结合形式的RalA、以及SERCA2。
特别地，上述方法进一步包括将已被确定为能够结合所述多肽的物质施用于所述信号通路的任一多肽或其片段、衍生物、同源物或突变体。特别地，上述方法中所述任一多肽或其片段、衍生物、同源物或突变体已经重组产生。特别地，上述方法进一步包括：确定所述测试物质是否能够替换Ral-GDP信号通路上的所述任一多肽或其片段，而不影响原信号通路的活性。
本申请还涉及一种定量或定性检测受试者SERCA2活性的方法，包括：提供与RalGAPα-RalA信号通路中蛋白质相互作用的物质；将所述与RalGAPα-RalA信号通路中蛋白质相互作用的物质受试者的细胞，体液或组织接触；以及评估受试者RalGAPα-RalA信号通路中蛋白质的含量。
特别地，本发明还提供可用作定量或定性检测SERCA2活性的诊断工具。例如，提供与RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物和/或GDP结合形式RalA相互作用的生物制品，例如抗原。这些生物制品可以是荧光标记的。样品可以与标记的生物制品一起孵育，过量的未结合的蛋白可以被洗去，然后可以评估该组织的荧光活性，其将指示存在RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物 和/或GDP结合形式RalA。由此，与RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物和/或GDP结合形式RalA相互作用的生物制品可以用于检测细胞，体液，组织或生物体中是否存在RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物和/或GDP结合形式RalA及其含量。检测到的RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物和/或GDP结合形式RalA量可以与SERCA2的活性相关。
特别地，本发明还包括用于检测样品中RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物和/或GDP结合形式RalA的水平的试剂盒，其包括至少一种与RalGAPα1和/或RalGAPα1/β复合物和/或RalGAPα2和/或RalGAPα2/β复合物和/或GDP结合形式RalA相互作用的生物制品，例如抗原，无论它是标记的还是未标记的，和至少一种与该生物制品结合的试剂，例如标记的抗体。试剂盒还可以包括适当的生物标准品和对照样品，可以将实验检测的结果与之进行比较。
本申请还涉及一种检测受试者RalGAPα-RalA信号通路活性的方法，包括：获取受试者的体液或组织；以及检测体液或组织的第一细胞的转运Ca2+能力。特别地，上述方法还包括：获取RalGAPα-RalA信号通路相关基因的核酸；将获取的核酸表达在第二细胞中；以及检测第二细胞的转运Ca2+能力。特别地，所述第二细胞为非洲爪蟾卵母细胞。
本申请还涉及一种诊断受试者心肌病的方法，包括：提供与RalGAPα-RalA信号通路中蛋白质相互作用的物质；将所述与RalGAPα-RalA信号通路中蛋白质相互作用的物质受试者的细胞，体液或组织接触；以及评估受试者RalGAPα-RalA信号通路中蛋白质的含量。
本申请还涉及一种诊断受试者心肌病的方法，包括：通过如上任一所述方法检测第一细胞或者第二细胞转运Ca 2+能力。
附图说明
下面，将结合附图对本发明的优选实施方式进行进一步详细的说明，其中：
图1A-I显示根据本发明的一个实施例的压力超负荷心脏中RalGAPα1复合物的表达趋势；
图2A-H显示根据本发明的一个实施例的RalGAPα1缺陷导致小鼠心脏功能障碍并加剧TAC诱导的心肌病；
图3A-J显示根据本发明的一个实施例的SERCA2为与RalGAPα1复合物相互作用的靶标，并且RalGAPα1复合物调节心肌细胞中的SR Ca 2+再摄取；
图4A-D显示根据本发明的一个实施例的GDP结合形式的RalA在RalGAPα1的下游调节SERCA2；
图5A-L显示根据本发明的一个实施例的RalGAPα1和RalA-GDP促进SERCA2a的寡聚化；
图6A-D显示根据本发明的一个实施例的GDP结合形式的RalA S28N突变体的表达针对TAC诱发的心肌病的保护；
图7显示根据本发明的一个实施例的RalGAPα1-RalA信号通路模型；以及
图8显示根据本发明的一个实施例的RalGAPα2缺陷导致新生大鼠原代心肌细胞中的SR Ca 2+再摄取障碍。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在以下的详细描述中，可以参看作为本申请一部分用来说明本申请的特定实施例的各个说明书附图。在附图中，相似的附图标记在不同图式中描述大体上类似的组件。本申请的各个特定实施例在以下进行了足够详细的描述，使得具备本领域相关知识和技术的普通技术人员能够实施本申请的技术方案。应当理解，还可以利用其它实施例或者对本申请的实施例进行结构、逻辑或者电性的改变。
术语“约”是指由用于获得测量的设备的典型错误率而造成的测量例如体积、时间、压力、浓度等的数值的变化。在一个实施方式中，术语“约”意味着在所记载数值的5％-10％以内；优选地，术语“约”意味着在所记载数值的3％-5％以内。
术语“心脏衰竭”是指以典型症状(例如呼吸困难、踝关节肿胀和疲劳)为特征的临床综合征。在某些情况下，心脏衰竭可能伴有由结构性和功能性心脏异常引起的表现(例如颈静脉压升高、肺裂音和外周性水肿)，导致心输出量降低和/或休息时或应激期间心脏内压力升高。
术语“急性心脏衰竭”或“AHF”在本文可互换使用，并且通常是指心脏衰竭的症状和/或征兆的快速发作或恶化，需要立即治疗和住院。
术语“慢性心脏衰竭”或“CHF”在本文中可互换使用，并且是指当前基于心脏衰竭征兆和症状的存在以及左心室射血分数(LVEF)的慢性心脏衰竭的临床分类。CHF可分为三类：“射血分数降低的心脏衰竭”或“HFrEF”，其特征是LVEF小于约40％；“具有中程射血分数的心脏衰竭”或“HFmEF”或“HFmrEF”，其特征是LVEF为约40％至约49％；和“射血分数保留的心脏衰竭”或“HFpEF”，其特征是LVEF等于或大于约50％。术语“HFmrEF”和“HFpEF”包括两个附加标准，即利尿钠肽水平升高(BNP>35pg/ml和/或NT-proBNP>125pg/mL)并伴有结构和/或功能性心脏病的证据(左心室肥大和/或左心房增大和/或舒张功能障碍的证据)。
术语“治疗”是指在疾病或病症的治疗或改善中成功的任何标志。治疗可以包括例如降低或减轻疾病或病症的一种或多种症状的严重性，或者可以包括降低个体例如人类患者所经历的疾病、缺陷、障碍或不利病症等的症状出现的频率。
术语“预防”是指在个体例如人类患者中预防疾病或病症。例如，如果具有发生心脏衰竭的风险的个体应用本发明的方法治疗后不发生心脏衰竭，则在该个体中所述疾病已得到预防。
术语“治疗或预防”有时在本文中用于指称导致疾病或病症的某种程度的治疗或改善的方法，并且设想了指向该目的的各种结果，包括但不限于所述病症的完全阻止。
术语“活性成分”是指物质被施用到的个体提供有益效果的物质。“治疗有效量”或“治疗有效剂量”是组合物或活性成分的足以为所述组合物或活性成分被施用到的个体提供有益效果的量。
术语“可药用载体”意味着可以与活性成分或包括活性成分的混合物组合并且在组合后可用于将所述化合物施用到哺乳动物的化学组合物。
术语“载体”将保护生物制品免于从体内迅速消除，以及将生物制品运送至功能区域。在本申请中，载体可将RalGAPα1、RalGAPα2、RalGAPα1/β复合物、、RalGAPα2/β复合物、RalA、GDP结合形式的RalA、RalA S28N或者GDP结合形式RalA S28N、SERCA2、SERCA2a寡聚体以及上述蛋白或核酸的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因运载至受试者体内。在本申请中，载体可以具有药物系统控缓释的特性，具有理想的释药速度和良好的缓控释效果。在本申请中，载体可以具有良好的生物相容性。生物降解性和生理性能。
术语“第一递送载体”主要为高分子生物材料，用于将蛋白质或多肽形式的生物制品，如包含RalGAPα1、RalGAPα2、RalGAPα1/β复合物、RalGAPα2/β 复合物、RalA、GDP结合形式的RalA、RalA S28N或者GDP结合形式RalA S28N、SERCA2、SERCA2a寡聚体以及上述蛋白的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因运载至受试者体内。其中，第一递送载体包括：植入物，脂质体、纳米粒、水凝胶、微球、微囊化递送系统，以及纳米生物医学载体等。
术语“第二递送载体”主要用于将核酸分子，如包含RalGAPα1、RalGAPα2、RalGAPα1/β复合物、RalGAPα2/β复合物、RalA、GDP结合形式的RalA、RalA S28N或者GDP结合形式RalA S28N、SERCA2、SERCA2a以及上述核酸的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列和同系物、同源基因的DNA、RNA等运载至受试者体内，并在受试者体内表达或表达后释放至受试者体内。其中，第二递送载体包括：腺相关病毒(AAV)、疱疹病毒、牛痘病毒、RNA病毒如逆转录病毒，胶体分散体系以及纳米生物医学载体等。
所述载体在一些实施例中，本发明的生物制品可以包括在第一递送载体和/或第二递送载体中。
在一些实施例中，第一递送载体为例如控释制剂，包括植入物和脂质体、纳米粒、水凝胶、微球、微囊化递送系统等。第一递送载体可以使用可生物降解的，生物相容的聚合物，例如聚酯类、聚丙烯酸酯类及其共聚物，如聚乳酸及其共聚物、聚-β-羟基羧酸酯、乙烯乙酸乙烯酯、聚酐、聚乙醇酸、胶原蛋白、聚原酸酯、由甲基丙烯酸、丙烯酸盐及丙烯酸酯如甲基丙烯酸丁酯、甲基丙烯酸丙二醇酯等单体聚合而成的聚丙烯酸等。制备这种制剂的方法对于本领域技术人员来说是已知的。在一些实施例中，第一递送载体为壳聚糖及其他多糖、藻酸盐、胶原、明胶、纤维蛋白、糖胺多糖、琼脂糖等。在一些实施例中，含有生物制品的脂质体悬浮液也可用作药学上可接受的载体。在一些实施例中，第一递送载体为纳米生物医学载体。
在一些实施例中，可将编码本发明生物制品的DNA导入细胞，然后在细 胞中表达由DNA编码的本发明的生物制品。在一些实施例中，本发明的生物制品的递送可使用第二递送载体实现。在一些实施例中，第二递送载体为重组表达载体如嵌合病毒或胶体分散系统或纳米生物医学载体。可用于将DNA导入细胞的各种病毒载体包括腺病毒、疱疹病毒、牛痘病毒或RNA病毒如逆转录病毒。在一些实施例中，胶体分散体系包括高分子复合物、纳米胶囊、微球、珠粒和脂质基体系。脂质基体系包括水包油乳液、胶束、混合胶束和脂质体。脂质体是可用作递送载体的人工膜囊泡。RNA、DNA或者完整的病毒粒子可以包封在含水的内部，并以生物活性形式递送到细胞。使用脂质体载体将基因有效转移到细胞中的方法是本领域已知的。脂质体的组合物通常包括磷脂的组合(典型地与类固醇，特别是胆固醇组合)，也可以使用其它磷脂或脂质。
术语“RalGAPα-RalA信号通路”是指RalGAPα蛋白通过与小G蛋白RalA相互作用，进而调控心肌细胞SERCA2的Ca 2+转导活性的信号通路。其中，RalGAPα蛋白可以是RalGAPα1或RalGAPα2。RalGAPα-RalA信号通路的蛋白质包括RalGAPα1、RalGAPα1/β复合物、RalGAPα2、RalGAPα2/β复合物、RalA、GDP结合形式的RalA、以及SERCA2。”
术语“RalGAPα1-RalA信号通路”是指RalGAPα1通过与小G蛋白RalA相互作用，进而调节心肌细胞SERCA2的Ca 2+转导活性的信号通路。其中，GDP结合形式的RalA可直接调节SERCA2的Ca 2+转导活性，而RalGAPα1与RalGAPβ结合而成的复合体可调节RalA的内在活性，进而调节SERCA2的Ca 2+转导活性。
术语“基序序列”是指是指DNA、蛋白质等生物大分子中的保守序列，介于二级和三级结构之间的另一种结构层次。
术语“同系物”是指结构相似、分子组成相差若干个"CH2"原子团的有机化合物。
术语“同源基因”是指一类含有同源框的基因。
术语“融合蛋白”有两种不同的含义，一种是通过DNA重组技术得到的两个基因重组后的表达产物。另一种含义就是介导两个细胞质膜融合的一组蛋白，
术语“表达盒”是指包含启动子，可以使得Ral S28N特异在心肌细胞中表达的盒子。其中除启动子及Ral S28N基因外，还包括运载该启动子和基因的载体等。
术语“复合物”是指两种或多种蛋白质的结合。这种结合可以是共价的或非共价的，包括例如复合物中两种蛋白质之间的离子、亲水性和疏水性相互作用。典型地，形成复合物的蛋白质彼此相互作用，使得鉴定或检测复合物中的第一蛋白质导致鉴定或检测与第一蛋白质形成复合物的一种或多种其它蛋白质。蛋白质复合物可以在体内鉴定，其中两种或多种蛋白质例如在细胞中自然地彼此结合以形成复合物。或者，可以在体外形成复合物，其中当将这些蛋白加入到相同的反应混合物中时，在两种或多种蛋白之间发生相互作用。
术语“RalGAPα1”、“RalGAPα2”、“RalGAPβ”、“RalA”、“RalB”、“GDP”以及“SERCA2”、“SERCA2a”均为序列已知且部分功能已知的基因，或基因表达的多肽、蛋白。其中，“RalGAPα1”序列见NCBI网站Gene ID:253959；“RalGAPα2”序列见NCBI网站Gene ID:5718；“RalGAPβ”序列见NCBI网站Gene ID:57148；“RalA”序列见NCBI网站Gene ID:5898；“RalB”序列见NCBI网站Gene ID:5899；以及“SERCA2”序列见NCBI网站Gene ID:488；“SERCA2a”序列见NCBI网站Gene ID:11938。SERCA2功能障碍是心脏衰竭的标志。因此，SERCA2功能的恢复是治疗这种疾病的有吸引力的策略。本发明基于发明人发现的RalGAPα1-RalA信号模型(参考图7)。RalGAPα1复合物与SERCA2相互作用，正向调节SERCA2的活性和SR Ca 2+再摄取。RalGAPα1复合物的下游调节因子，GDP结合状态RalA决定了与SERCA2动态的相互作用。这一信号模型对于保护心脏功能至关重要。
由此，本发明提出利用RalGAPα1-RalA信号模型提供的调控SERCA2活性的方式预防和治疗心肌病或心脏衰竭药物。在一些实施例中，这样的药物可以是生物制品，包括：蛋白、蛋白复合物、多肽和抗体。如上所述，本发明的生物制品靶向心肌细胞中包含SERCA2的RalGAPα1-RalA信号通路。
在一些实施例中，这些药物包括RalGAP蛋白复合物，特别是催化亚基RalGAPα1与调控亚基RalGAPβ形成的复合物RalGAPα1/β，即RalGAPα1复合物，催化亚基RalGAPα2与调控亚基RalGAPβ形成的复合物RalGAPα2/β，即RalGAPα2复合物，以及RalGAPα1复合物的剪接变体，RalGAPα2复合物的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等。在一些实施例中，这些药物包括RalGAPα1、RalGAPα2、RalGAPα1或RalGAPα2的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等。在一些实施例中，这些药物包括RalA、GDP结合形式的RalA，以及RalA、GDP结合形式的RalA的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等。例如：RalA S28N突变体。
在一些实施例中，本发明提供了包含本发明生物制品的组合物。这些组合物可适用于药物用途和对患者的给药。该组合物通常含有一种或多种本发明生物制品和药学上可接受的赋形剂。本文所用短语“药学上可接受的赋形剂”包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗菌剂和抗真菌剂、等渗剂和吸收延迟剂等。这种介质和试剂用于药物活性物质的用途在本领域中是已知的。本发明生物制品的组合物还可包含提供补充，附加或增强治疗功能的其它活性化合物。
在一些实施例中，本发明的生物制品可以包括在载体中，所述载体将保护生物制品免于从体内迅速消除，例如控释制剂，包括植入物和微囊化递送系统。可以使用可生物降解的，生物相容的聚合物，例如乙烯乙酸乙烯酯，聚酐，聚 乙醇酸，胶原蛋白，聚原酸酯和聚乳酸。制备这种制剂的方法对于本领域技术人员来说是已知的。在一些实施例中，含有生物制品的脂质体悬浮液也可用作药学上可接受的载体。
在一些实施例中，可将编码本发明生物制品的DNA导入细胞，然后在细胞中表达由DNA编码的本发明的生物制品。在一些实施例中，本发明的生物制品的递送可使用重组表达载体如嵌合病毒或胶体分散系统来实现。可用于将DNA导入细胞的各种病毒载体包括腺病毒、疱疹病毒、牛痘病毒或RNA病毒如逆转录病毒。在一些实施例中，胶体分散体系包括高分子复合物、纳米胶囊、微球、珠粒和脂质基体系。脂质基体系包括水包油乳液、胶束、混合胶束和脂质体。脂质体是可用作递送载体的人工膜囊泡。RNA、DNA或者完整的病毒粒子可以包封在含水的内部，并以生物活性形式递送到细胞。使用脂质体载体将基因有效转移到细胞中的方法是本领域已知的。脂质体的组合物通常包括磷脂的组合(典型地与类固醇，特别是胆固醇组合)，也可以使用其它磷脂或脂质。
特别地，在某些实施例中，本发明的生物制品为RalA S28N重组蛋白或者编码RalA S28N重组蛋白的DNA分子，其用于在心肌细胞中表达或过表达RalA S28N重组蛋白。
特别地，在某些实施例中，本发明的生物制品为腺相关病毒(AAV)系统中使用Ctnt启动子驱动的用于表达RalA S28N突变体的表达盒。通过静脉注射用于表达RalA S28N突变体的AAV病毒，能够在心脏中特异性表达RalA S28N突变体。
RalGAPα1复合物/RalGAPα2复合物在压力超负荷引起的心肌病或心脏衰竭中发挥保护作用。RalGAPα1复合物/RalGAPα2复合物能够正向调节SERCA2活性，加速SR Ca 2+再摄入。同样地，作为RalGAPα1复合物的下游调控因子，GDP结合形式的RalA也具有相同的功能。本申请所说的心肌病或 心脏衰竭是由SERCA2介导的钙离子转运异常表征。在一些实施例中，其中心肌病是心脏衰竭。在一些实施例中，其中心肌病是慢性心脏衰竭。
进一步地，本发明提出了正向调节心肌细胞中SERCA2活性的方法和用途，特别是，正向调节心肌细胞中SERCA2的SR Ca 2+再摄入的方法和用途。在一些实施例中，这些方法和用途包括给予人受试者有效量的本发明的生物制剂，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式的RalA或者其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因和/或GDP结合形式的RalA或者其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等。在一些实施例中，以上这些方法和用途还包括将编码本发明的生物制品，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA或其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等的DNA分子，导入心肌细胞中。
受磷蛋白是众所周知的SERCA2调节剂，与Ca 2+泵结合并施加抑制作用。与受磷蛋白的负向调节相反，RalA在与Ca 2+泵结合后对SERCA2施加正向调节。受磷蛋白和RalA都受到严格调控，并表现出与SERCA2的动态相互作用。
由此，本发明还提出通过调节SERCA2的功能调控心肌细胞中Ca 2+的胞内平衡的方法和用途。通过给予心肌细胞有效量的GDP结合形式的RalA或者受磷蛋白，能够通过与SERCA2的相互作用动态地正向或反向调节Ca 2+的胞内平衡。在一些实施例中，以上的方法和用途包括给予人受试者有效量的本发明的生物制剂，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或RalA和/或GDP结合形式的RalA或者其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、 同源基因等。在一些实施例中，以上这些方法和用途还包括将编码本发明的生物制品，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或RalA和/或GDP结合形式RalA或其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等的DNA分子，如RalA S28N或者GDP结合形式RalA S28N，导入心肌细胞中。
GDP结合形式的RalA引起SERCA2的寡聚化，增加SERCA2的Ca 2+转运活性但不增加其ATPase活性。然而，这种与GDP结合形式RalA的相互作用改变SERCA2配置以促进Ca 2+泵的寡聚化并不依赖于Thr 484的磷酸化。
由此，本发明还涉及正向调节SERCA2a寡聚化的方法和用途。通过给予心肌细胞有效量的GDP结合形式的RalA正向调节SERCA2a寡聚化。在一些实施例中，这些方法和用途包括给予人受试者有效量的式I化合物或含有式I化合物的组合物；或者给予人受试者有效量的本发明的生物制剂，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式的RalA或者其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等。在一些实施例中，以上这些方法和用途还包括将编码本发明的生物制品，例如RalGAPα1和/或RalGAPα1复合物和/或GDP结合形式RalA或其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等的DNA分子，如RalA S28N或者GDP结合形式RalA S28N，导入心肌细胞中。
RalGAPα-RalA信号通道模型通过调节SERCA2发挥作用，对Ca 2+胞内平衡和维护心脏的功能至关重要，其不但在压力超负荷引起的心脏衰竭中发挥保护作用，对治疗继发的心脏衰竭也具有预防和治疗意义。由此，本发明还提出利用RalGAPα1复合物和/或RalGAPα2复合物和/或GDP结合形式的RalA预防和治疗心肌病或心脏衰竭的方法和用途。在一些实施例中，这些方法和用 途包括给予人受试者有效量的本发明的生物制剂，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式的RalA或者其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等。在一些实施例中，以上这些方法和用途还包括将编码本发明的生物制品，例如RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA或其剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白，同系物、同源基因等的DNA分子，导入心肌细胞中。
在一些实施例中，本发明提供正向调节心肌细胞中SERCA2活性的方法和用途；特别地，正向调节心肌细胞中SERCA2的SR Ca 2+再摄入的方法和用途。进一步地，在一些实施例中，本发明还提供通过调节SERCA2的功能调控心肌细胞中Ca 2+的胞内平衡。在一些实施例中，本发明还提供了正向调节SERCA2a寡聚化的方法和用途。在一些实施例中，本发明提供利用预防和治疗心肌病或心脏衰竭的方法和用途。
在一些实施例中，本申请正向调节SERCA2a寡聚化的方法，包括：给予受试者有效量的如上任一药物组合物。在一些实施例中，本申请正向调节SERCA2活性的方法，包括：给予受试者有效量的如上任一药物组合物。在一些实施例中，本申请预防和治疗受试者心肌病的方法，包括：给予受试者有效量的如上任一药物组合物。在一些实施例中，其中心肌病是SERCA2介导的钙离子转运异常引起。其中所述心肌病是心脏衰竭，进一步地，心肌病是慢性心脏衰竭。
本发明还提供可用作定量或定性检测SERCA2活性的诊断工具。例如，提供与RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或RalAGDP和/或结合形式RalA相互作用的生物制品，例如抗原。这些生物制品可以是荧光标记的。样品可以与标记的生物制品一起孵育，过量的未 结合的蛋白可以被洗去，然后可以评估该组织的荧光活性，其将指示存在RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA。由此，与RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA相互作用的生物制品可以用于检测细胞，体液，组织或生物体中是否存在RalGAPα1复合物和/或RalGAPα2复合物和/或GDP结合形式RalA及其含量。检测到的RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA量可以与SERCA2的活性相关。
本发明还包括用于检测样品中RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA的水平的试剂盒，其包括至少一种与RalGAPα1和/或RalGAPα1复合物和/或RalGAPα2和/或RalGAPα2复合物和/或GDP结合形式RalA相互作用的生物制品，例如抗原，无论它是标记的还是未标记的，和至少一种与该生物制品结合的试剂，例如标记的抗体。试剂盒还可以包括适当的生物标准品和对照样品，可以将实验检测的结果与之进行比较。
在一些实施例中，定量或定性检测受试者SERCA2活性的方法，包括：提供与RalGAPα-RalA信号通路中蛋白质相互作用的物质，该物质可以是基因、蛋白质、多肽，也可以是有机化合物、化合物组合物等。将该物质受试者的细胞，体液或组织接触，触发该物质与RalGAPα-RalA信号通路中任何一个蛋白、多肽或基因的互作。评估受试者RalGAPα-RalA信号通路中蛋白质的含量。其中被评估的蛋白质可以是RalGAPα-RalA信号通路上的任何一种蛋白。
在一些实施例中，检测受试者RalGAPα-RalA信号通路活性的方法，包括：获取受试者的体液或组织，该体液或组织中包括RalGAPα-RalA信号通路中的全部组成部分。若该受试者心肌健康，则获取的体液或组织的细胞具有正常的钙离子转运活性；或该受试者患有心肌疾病，如心衰，则获取的体液或组织的 细胞不能正常转运钙离子。检测体液或组织的第一细胞的转运Ca 2+能力。其中，第一细胞为获取的受试者体液或者组织中的细胞。
在一些实施例中，检测受试者RalGAPα-RalA信号通路活性，还包括获取RalGAPα-RalA信号通路相关基因的核酸；并将获取的核酸表达在第二细胞中。其中第二细胞为可以体外表达基因的活细胞，如非洲爪蟾卵母细胞。检测第二细胞的转运Ca 2+能力。
在一些实施例中，可以使用电生理技术检测第一细胞或第二细胞转运钙离子的能力。在一些实施例中，可以使用膜片钳技术检测第一细胞或第二细胞转运钙离子的能力。
在一些实施例中，诊断受试者心肌病的方法，包括：提供与RalGAPα-RalA信号通路中蛋白质相互作用的物质；将该物质受试者的细胞，体液或组织接触；评估受试者RalGAPα-RalA信号通路中蛋白质的含量。
在一些实施例中，可以通过检测、评估RalGAPα-RalA信号通路在第一细胞或者第二细胞中转运Ca 2+能力诊断受试者是否患有心肌病。
实施例1：压力超负荷心脏中RalGAPα1复合物的表达增加
本实施例中通过观察两种RalGAP复合物及其下游的Ral小G蛋白在压力超负荷心脏中的表达，发现它们在高血压诱发的心肌病中所起的作用。
本实施例及以后实施例中所进行的蛋白组学方法可以按照如下的步骤进行。本实施例及以后实施例中使用的商业抗体参见如下表1：
抗体名称 公司名称 试剂盒
anti-RalA CST #4799
anti-HA CST #3724
anti-RalB Abclonal WH079944
anti-β-MHC Sigma t9283
anti-Flag Sigma F9291
anti-cTnT Thermo Fisher MA5-12960
anti-DDR2 Santa Cruz Biotechnology sc-8989
anti-GFP Santa Cruz Biotechnology sc-9996
anti-GST Abclonal AE001
anti-SERCA2a Proteintech 13985-1-AP
GAPDH Proteintech 60004-1
本实施例及以后实施例中用于目标基因的QPCR的引物参见如下表2：
基因名称 正向引物 反向引物
RalGAPα1-Mus 5′-AGATCAGACGGGAAGGTGTT-3′ 5′-CTTGAAGGCTGAGTGGAGGA-3′
RalGAPα2-Mus 5′-CAGGAGTGGAGAAGGCAAGA-3′ 5′-TGGGGCTGTAACCTTGAGAG-3′
RalGAPβ-Mus 5′-AAATCCAAGGAGCCACTGGA-3′ 5′-GGCTCCAACTGCTTATTCCG-3′
RalGAPα1-Rat 5′-CCAGCACCACTTAGAGCCAA-3′ 5′-GCGGCTTTTGCTAGTTCGAG-3′
RalGAPα2-Rat 5′-GTTCAGGTGAAATGGATCCTGC-3′ 5′-TTCGGATGAGACCTCCTTGG-3′
RalGAPβ-Rat 5′-CCAGCTTATTTATCCAGCGTTATTC-3′ 5′-ATGAGGGAGGGGCAACAAAG-3′
RalA 5′-ACAGGATGGCTGCAAACAAG-3′ 5′-TGAACTGCAGAGTCAGAGCA-3′
RalB 5′-CTTTCCCTCCTCAACACCCT-3′ 5′-AGCCTTCCCTTCATCTGCTT-3′
β-MHC 5′-ACCCCTACGATTATGCG-3′ 5′-GTGACGTACTCGTTGCC-3′
Rcan1.4 5′-GTGTGGCAAACGATGATGTC-3′ 5′-AGGAACTCGGTCTTGTGCAG3′
ANP 5′-TCGTCTTGGCCTTTTGGCT-3′ 5′-TCCAGGTGGTCTAGCAGGTTCT-3′
BNP 5′-AAGCTGCTGGAGCTGATAAGA-3′ 5′-GTTACAGCCCAAACGACTGAC-3′
CollA1 5′-GGAGAGAGCATGACCGATGG-3′ 5′-AAGTTCCGGTGTGACTCGTG-3′
Col3A1 5′-CCCAGAACATTACATACCA-3′ 5′-GATTAAAACAAGATGAACAC-3′
Serca2a 5′-ACTTCTTGATCCTCTACGTG-3′ 5′-AAATGGTTTAGGAAGCGGTT-3′
Pmcal 5′-TTAGTCTGGGAAGCATTACAAGATGTCAC-3′ 5′-CTTCTTCCCCAACAGAAACTTCTCC-3′
Pmca4 5′-ACGTCTTCCCACCCAAGGTTC-3′ 5′-CCAGCAGCCCACACTCTGTC-3′
Ncx 5′-GATCATCCGATTCCCTCTACTG-3′ 5′-GTCAGTGGCTGCTTGTCATC-3′
Ryr2 5′-TCAAACCACGAACACATTGAGG-3′ 5′-AGGCGGTAAAACATGATGTCAG-3′
Ltcc 5′-CAATGGTCAATGAAAACACGA-3′ 5′-GGCTCCCATAGTTGGAACCT-3′
RalA-AAV 5′-TACGATGAGTTTGTGGAGGACT-3′ 5′-CTCCTGCCCAGCTGTATCTAAG-3′
36B4-Mus 5′-TAAAGACTGGAGACAAGGTG-3′ 5′-GTGTACTCAGTCTCCACAGA-3′
36B4-Rat 5′-TCCAGAGGTACCATTGAAATCC-3′ 5′-GTAGATGCTGCCATTGTCAAAC-3′
本实施例和其后实施例中使用的所有小鼠都在特定的无病原体条件下，以 光/暗周期为12小时饲喂。除非特别说明，小鼠可以在笼子里自由获取食物和水。通过对麻醉的雄性小鼠(2-3个月大)进行TAC手术构建压力超负荷的心脏。具体而言，在小鼠腹膜内注射氯胺酮(100mg/kg)和甲苯噻嗪(10mg/kg)。对于实验组，通过手术暴露足弓主动脉并使用27号针头用6-0缝线结扎以产生一个直径为0.413毫米的收缩。对于作为对照的假手术组，小鼠接受相同的外科手术而只是不进行结扎。
随后，小鼠被处死以分离心脏组织。在裂解前，将小鼠心脏组织在液氮中速冻并储存在零下80℃。然后，在裂解缓冲液进行均质化再在冰上裂解30分钟，通过离心去除组织碎片获得组织裂解物。
通过用抗体偶联蛋白G-Sepharose或GFP结合剂在4℃下保持16小时进行目标蛋白的免疫沉淀。清洗树脂以去除非特异性结合蛋白，然后在SDS样品缓冲液中洗脱免疫沉淀复合物。
裂解物或免疫沉淀物被电泳分离。电泳后，分离的蛋白质被免疫印迹到硝酸纤维素膜与一抗孵育。在使用辣根过氧化物酶(HRP)偶联的二抗进一步探测后，膜与HRP底物一起孵育以增强化学发光。
免疫沉淀复合物通过SDS-PAGE分离并通过考马斯蓝染色进行可视化。然后，在凝胶上切下感兴趣的蛋白质条带，并使用胰蛋白酶作为消化酶进行凝胶内消化，然后通过LC-MS/MS分析每个凝胶条带。
使用
Figure PCTCN2022127868-appb-000001
试剂从中提取总RNA，并使用
Figure PCTCN2022127868-appb-000002
RT试剂盒逆转录成cDNA。使用Roche Lightcycler Real-Time PCR系统和表2中列出的引物对感兴趣的基因进行QPCR分析。使用Prism软件通过两组的t检验或通过单向或双向方差分析对多组进行数据分析。在p<0.05时，差异被认为具有统计学意义。
本申请的发明人发现，压力超负荷心脏中在蛋白质水平上βMHC增加(参见图1A)。在假手术和TAC手术的心脏中Ralgapα1和Ralgapβ的mRNA水 平相当而在接受TAC手术的心脏中二者的蛋白质水平显着增加(参见图1A-C)。相反，在TAC手术的心脏中，RalGAPα2的mRNA和蛋白质水平都没有变化(参见图1A-C)。在TAC手术的心脏中，RalGAP复合物下游的RalA和RalB在mRNA和蛋白质水平上都保持正常(参见图1A-C)。
血管紧张素II(Ang-II)和去甲肾上腺素(NE)是压力超负荷诱导的心肌病的神经激素的两个关键调节因子。NE治疗增加了原代新生大鼠心室心肌细胞(NRVC)中的RalGAPα1蛋白，但不增加其mRNA水平(参见图1D-F)。类似地，Ang-II刺激会增加其在初级NRVC中的蛋白质水平(参见图1G-I)。总之，这些数据表明RalGAPα1复合物在压力超负荷诱导的心肌病中起重要作用。
实施例2：RalGAPα1缺陷导致心脏功能障碍并加剧TAC诱导的心肌病
心肌细胞和成纤维细胞是心脏中的两个细胞群。尽管都存在于心脏中，RalGAPα1和α2都优先在心肌细胞中表达，而不是在心脏的成纤维细胞中表达(参见图2A)。在本实施例中，为了发现RalGAPα1在调节心脏功能中的作用，发明人生成了RalGAPα1心肌细胞特异性缺失小鼠模型(RalGAPα1-cKO)。
RalGAPα1 f/f小鼠(其获得可参考文献：Chen Q,Rong P,Zhu S,Yang X,Ouyang Q,Wang HY,Chen S.Targeting ralgapalpha1in skeletal muscle to simultaneously improve postprandial glucose and lipid control.Sci Adv.2019；5:eaav4116)与αMHC-Cre小鼠(其获得可参考文献：Agah R,Frenkel PA,French BA,Michael LH,Overbeek PA,Schneider MD.Gene recombination in postmitotic cells.Targeted expression of cre recombinase provokes cardiac-restricted,site-specific rearrangement in adult ventricular muscle in vivo.J Clin Invest.1997；100:169-179)用于产生心肌细胞特异性RalGAPα1敲除小鼠(RalGAPα1-cKO)。RalGAPα1 f/f X RalGAPα1 f/f-Cre交配被设置为产生RalGAPα1 f/f(对照小鼠)和RalGAPα1 f/f-Cre(RalGAPα1-cKO小鼠)。RalGAPα1 f/f小鼠使用以下引物进行基因分型：
5’-GAGATGGCGCAACGCAATTAATG-3’和
5’-GGCTGCAAAGAGTAGGTAAAGTGCC-3’。
Cre小鼠使用以下引物进行基因分型：
5’-GCCTGCATTACCGGTCGATGC-3’和
5’-CAGGGTGTTATAAGCAATCCC-3’。
同样地，对于上述小鼠心肌细胞进行蛋白质组学分析。进一步地，采用以下方式以获得小鼠心脏切片的图像：小鼠被处死以分离心脏。然后，在4℃下将心脏固定在4％PFA中过夜，并嵌入石蜡中。然后，使用Leica RM2016切片机将心脏切成5μm厚的切片。对心脏切片进行苏木精-伊红染色，并使用Olympus BX53F显微镜拍照。
正如预期的那样，通过将RalGAPα1 f/f与αMHC-Cre小鼠交配，RalGAPα1在心脏中减少，但在其他组织(包括骨骼肌、肝脏、棕色脂肪组织和白色脂肪组织)并未改变(参见图2B)。心肌细胞的RalGAPα1特异性缺失不影响RalGAPα2的表达(参见图2B)。由于RalGAPβ的稳定性取决于RalGAPα，RalGAPβ在心脏中表现出适度减少，但在其他组织中保持正常(参见图2B)。这些数据表明，RalGAPα1-cKO小鼠及其衍生的心肌细胞适合研究RalGAPα1在心脏中的功能。
有趣的是，发明人发现心脏中RalGAPα1的缺陷会损害小鼠的心脏功能。RalGAPα1-cKO小鼠的射血分数(EF)和短轴缩短率(FS)均显著低于RalGAPα1 f/f同窝小鼠(参见图2C)。将RalGAPα1-cKO和RalGAPα1 f/f小鼠进行TAC手术。非常重要的发现是手术后RalGAPα1-cKO小鼠的死亡率明显高于RalGAPα1 f/f同窝小鼠(参见图2D)；同样，RalGAPα1-cKO小鼠的EF和FS也均低于对照小鼠(参见图2E-F)。随着心脏纤维化水平上升和心脏衰竭标志物(如Anp、Bnp、Col1a1和Col3a1)在RalGAPα1-cKO心脏中表达的增加(参见图2G-H)，与对照心脏相比，RalGAPα1的缺陷加剧了TAC诱导的心脏重构。这些数据表明RalGAPα1复合物的上调在压力超负荷的心脏中 起着重要的保护作用。
实施例3：SERCA2为与RalGAPα1复合物相互作用的靶标，并且RalGAPα1复合物调节心肌细胞中的SR Ca 2+再摄取
在本实施例中，采用蛋白质组学方法来鉴定RalGAPα1复合物相互作用的蛋白质，以明确RalGAPα1复合物调节心脏功能的机制。在HEK293细胞中表达GFP-RalGAPα1和HA-RalGAPβ，随后使用GFP-Trap珠对其进行免疫沉淀。通过质谱法鉴定与GFP-RalGAPα1免疫共沉淀的蛋白质。
人胚肾HEK293细胞购自中国医学科学院和北京协和医学院细胞资源中心(中国)，维持在含有10％(v/v)胎牛血清的DMEM培养基中，定期检测支原体。使用脂质体3000试剂(Thermo Fisher Scientific)进行细胞转染。
使用基于胶原酶方法分离原代小鼠心肌细胞。用胶原酶灌注肝素处理小鼠的心脏。胶原酶消化后，通过细胞过滤器(100μm筛孔)过滤细胞悬液以去除组织碎片。所得心肌细胞在Krebs-Henseleit缓冲液B中冲洗3次。
原代新生大鼠心肌细胞获自新生Sprague Dawley大鼠(出生后第0-3天)的心室。从新生大鼠中分离出心室并切成方块。用胰蛋白酶对心室方块进行顺序消化。通过细胞过滤器(70μm网孔大小)过滤细胞悬液以去除组织碎片。将得到的细胞接种在含有10％(v/v)胎牛血清的DMEM中1小时以允许成纤维细胞沉降以去除。然后，将心肌细胞在新鲜的补充有10％(v/v)胎牛血清的DMEM中制片。原代新生心肌细胞使用脂质体3000试剂(Thermo Fisher Scientific)转染。
基于Fluo-4-AM的方法测量心肌细胞中的钙瞬变。将心肌细胞培养在含有1mM的Hanks缓冲液中。加载Fluo-4-AM，利用GRASS S48刺激器使心肌细胞受到电刺激。使用蔡司LSM880共聚焦显微镜拍摄线扫描图像，并用IDL5.5(哈里斯地理空间解决方案)分析；其中，从钙瞬变峰值到衰落阶段从峰值到基础水平的63％的时间定义为衰减时间。
采用以下方法获得HEK293细胞中的钙成像：针对表达SERCA2a的HEK293细胞加载5μM Fluo-4-AM，然后用100μM ATP刺激。使用Olympus拍摄细胞的帧扫描图像。使用基于Di-8-ANEPPS的成像方法分析心肌细胞中t小管(TT)组织。将分离的原代心肌细胞用Di-8-ANEPPS染色。使用Carl Zeiss880共聚焦显微镜获得细胞图像，并且对获得的图像进行快速傅立叶变换。在细胞图像的傅立叶光谱中使用ImageJ软件确定TT功率(峰值振幅)。
进一步地，分离含有粗制SR膜囊泡的微粒体以用于检测微粒体中SERCA2-ATPase活性。通过在含有100mM KCl、10mM HEPES(pH 7.4)、5mM MgCl2、100μM CaCl2、1.5mM ATP、2μM A23187和5mM叠氮化钠的测定缓冲液孵育微粒体(50μg蛋白质)进行ATP水解反应。在30℃下保持30分钟，并通过加入冰冷的10％TCA终止。水解反应是在不存在或存在5μM毒胡萝卜素的情况下进行，以分别确定总的和对毒胡萝卜素不敏感的钙泵数量。通过在总活性中减去毒胡萝卜素不敏感的Ca 2+-ATPase活性来确定对毒胡萝卜素敏感的活性Ca 2+-ATPase(SERCA2-ATPase)。
进一步地，分离含有粗制SR膜囊泡的微粒体以检测微粒体中Ca 2+的摄取。使用基于Fura-2的方法测量Ca 2+摄取。微粒体在含有2μM Fura-2游离酸的测定缓冲液(100mM KCl、10mM HEPES-KOH(pH 7.4)、10mM草酸盐、5mM MgCl2和10μM钌红)中被重新悬浮。添加ATP(5mM)和Ca 2+(2μM)以启动将Fura-2摄取到微粒体中。在340nM和380nM进行双激发，并使用荧光酶标仪在510nM记录发射的荧光。使用Clampfit 10.4(Molecular Devices)对游离Ca 2+作图，并使用开始吸收后曲线的线性部分计算Ca 2+摄取速率。
正如预期的那样，在免疫沉淀物中发现作为RalGAPα1的已知结合物RalGAPβ(参见图3A)。有趣的是，心脏Ca 2+胞内平衡的关键调节因子SERCA2也被确定为RalGAPα1的潜在相互作用标靶(参见图3A)。通过蛋白质印迹免疫沉淀进一步验证了GFP-RalGAPα1免疫沉淀物中SERCA2的存在(参见图 3B)。并且，内源性SERCA2与来自心脏裂解物免疫沉淀的内源性RalGAPα1免疫共沉淀(参见图3C)。
在HEK293细胞中过表达RalGAPα1，并测量了由添加ATP引起的Ca 2+瞬变。Ca 2+瞬变的半峰全持续时间(FDHM)和时间常数Tau是反映Ca 2+重新摄取到ER中速率的两个量度，峰值是细胞溶质中Ca 2+的量度。RalGAPα1的过表达导致HEK293细胞中Ca 2+瞬变的FDHM和Tau显着降低，表明Ca 2+加速重新摄取进入ER(参见图3D)。在过表达RalGAPα1的细胞中，Ca 2+瞬变的峰值显着增加，表明细胞溶质中Ca 2+的减少(参见图3D)。然后，使用小干扰RNA(siRNA)敲低NRVC中的RalGAPα1，并检查RalGAPα1减少对Ca 2+瞬变的影响。结果发现，Ca 2+瞬变的FDHM和Tau显着增加，而它们的峰值在RalGAPα1减少的NRVC中被压低(参见图3E)。因此，发明人发现，RalGAPα1复合物与SERCA2的相互作用表明其通过控制SR Ca 2+再摄取来调节Ca 2+胞内平衡。
心肌细胞RalGAPα1的特异性缺陷不影响心脏中Ltcc、Ncx和Ryr2的mRNA水平。在RalGAPα1-cKO小鼠的心脏中，SERCA2a的表达在mRNA和蛋白质水平上也都是正常的。分离原代心肌细胞并分析这些细胞中的Ca 2+胞内平衡。t小管(TT)是心肌细胞中的内陷肌膜，其中含有富含离子通道和转运蛋白的膜微区。TT形成一个分支和互连的网络，它们的规律性在RalGAPα1缺陷细胞和对照细胞之间大体相当。测量由电刺激引起的原代心肌细胞中的Ca 2+瞬变时发现，在RalGAPα1缺陷的心肌细胞中，Ca 2+瞬变的FDHM和Tau显着增加(参见图3F-G)。如Ca 2+瞬变幅度的减小所证明的，这种Ca 2+重新摄取到SR的延长导致细胞溶质Ca 2+升高(参见图3F-G)。细胞溶质Ca 2+是通过钙调神经磷酸酶-NAFT途径表达Rcan1.4的诱导剂。与细胞溶质Ca 2+升高一致，与RalGAPα1 f/f对照心脏相比，RalGAPα1-cKO心脏中的Rcan1.4表达显着增加(参见图3H)。与对照细胞相比，RalGAPα1缺 陷的心肌细胞中Ca 2+的循环频率基本没有变化，这表明RalGAPα1缺陷可能不会影响从SR中自发的Ca 2+释放。与Ca 2+瞬时测定中延长的Ca 2+再摄取一致，与对照相比，SERCA2-ATPase活性和SR Ca 2+转运在从RalGAPα1缺陷的心脏分离出来微粒体中均显着降低(参见图3I-J)。
这些数据表明RalGAPα1通过调节SERCA2介导的SR Ca 2+再摄取来调控心肌细胞中的Ca 2+胞内平衡。RalGAPα1的缺陷延长了心肌细胞中SR Ca 2+的再摄取，这是RalGAPα1-cKO小鼠心脏功能障碍的基础。
实施例4：GDP结合形式的RalA在RalGAPα1的下游调节SERCA2
在本实施例中，进一步还分析了GDP结合的RalA与SERCA2的相互作用。RalGAPα1包括具有Asn 1949为活性关键残基的功能性GAP结构域。当引入Asn 1949Lys突变以使其GAP活性失活时，RalGAPα1在Ca 2+瞬态分析中失去了加速Ca 2+再摄取到ER中的能力。与野生型RalGAPα1相比，RalGAPα1的Asn 1949Lys突变体既不能降低FDHM和Tau，也不能增加Ca 2+瞬变的幅度(参见图3D)。对于RalA和RalB，RalGAPα1将这两种小G蛋白从GTP结合形式转化为GDP结合形式。发明人发现，当在HEK293细胞中共表达时，RalA而不是RalB与SERCA2相互作用(参见图4A)。令人惊奇的是，当在细胞中共表达时，GDP结合的RalA S28N突变体而不是GTP结合的RalA G23V突变体能够与SERCA2相互作用(参见图4A)。RalA的表达在RalGAPα1-cKO和RalGAPα1 f/f小鼠的心脏之间大致相当，而RalA的GTP结合形式在RalGAPα1缺陷的心脏中如所预期地增加(参见图4B)。
因此，RalA的GDP结合形式在RalGAPα1缺陷的心脏中可能减少。更重要的是，RalA S28N突变体的表达，而非RalA G23V突变体的表达，降低了FDHM和Tau，并增加了HEK293细胞(图4C)或原代新生大鼠心肌细胞(图4D)中Ca 2+瞬变的幅度。这表明RalA的GDP形式能够加速Ca 2+重新摄取到SR。这些数据表明RalGAPα1通过RalA的GDP结合形式调节 SERCA2a。
实施例5：RalGAPα1和RalA-GDP促进SERCA2a的寡聚化
在本实施例中，进一步研究了SERCA2a的寡聚化。如本领域技术人员所知，SERCA2a在单体和寡聚体之间切换，寡聚化能够增强其转运Ca 2+的活性。有趣的是，SDS凝胶的可视化印迹显示，当其与HEK293细胞中的RalGAPα1/β复合物共表达时，SERCA2a的寡聚化显著增加(参见图5A)。SERCA2a的寡聚化在RalGAPα1-cKO心脏中则显著降低(参见图5B)。SERCA2a寡聚化的减少不是由于Thr484磷酸化引起的。在RalGAPα1-cKO心脏中，上游激酶SPEG也正常表达而并未改变。RalGAPα1对SERCA2a和寡聚化的影响由下游靶标RalA介导。当通过shRNA敲低细胞中的RalA时，RalGAPα1与SERCA2a的相互作用和RalGAPα1诱导的SERCA2a寡聚化均被阻止(参见图5C-D)。RalA S28N突变体的表达，而不是RalA G23V突变体的表达，提高了高分子量SERCA2a(～300kDa)的比例和促进了两个SERCA2a单体之间的相互作用(参见图5E-F)。
与RalGAPα1-cKO心脏中SERCA2a的功能受损(参见图3I-J)相一致，RalGAPα1的过表达增强了HEK293细胞中Ca 2+泵的ATP酶水平和转运活性(参见图5G-H)。与RalA G23V突变体相比，RalA S28N突变体的表达显著增加了HEK293细胞中SERCA2a的Ca 2+转运活性，但不影响其ATPase活性(参见图5I-J)。
进一步地，本实施例研究了RalA是否对SERCA2a产生直接影响以调节其Ca 2+转运活性。在本实施例中，从大肠杆菌中表达并纯化了GST-RalA S28N重组蛋白，并发现它可以在GST-折叠测定中与Flag-SERCA2a相互作用(参见图5K)。重要的是，当将这种GST-RalA S28N重组蛋白添加到从表达Flag-SERCA2a的HEK293细胞纯化的微粒体中时，该GST-RalA S28N重组蛋白增加了SERCA2a的Ca 2+转运活性(参见图5L)。这表明RalA直接激活 SERCA2a的Ca 2+转运。
这些数据表明RalGAPα1通过与GDP结合的RalA促进SERCA2寡聚化，以增强Ca 2+泵的转运活动。RalGAPα1还可以通过与Ca 2+泵的直接相互作用来调节SERCA2a的ATPase活性。
实施例6：GDP结合形式的RalA S28N突变体的表达针对TAC诱发的心肌病的保护
在本实施例中，通过实例证明了GDP结合形式的RalA的升高对治疗压力超负荷诱发的心肌病具有明确的治疗价值。在本实施例发现，RalA S28N突变体的表达恢复RalGAPα1减少的NRVC中的Ca 2+胞内平衡。事实上，RalA S28N突变蛋白的表达逆转了RalGAPα1减少诱导的FDHM和Tau的增加，并缓解了由NRVC中的RalGAPα1缺陷引起Ca 2+瞬态峰的压低(图6A)。
进一步地，在本实施例中构建了在腺相关病毒(AAV)系统中使用Ctnt启动子驱动的表达盒以在心脏中特异性表达RalA S28N突变体。通过骨骼肌和肝脏中mRNA和蛋白质水平上的分析，本实施例证实RalA S28N突变体仅在心脏中表达，而不在其他组织中表达(参见图6B)。在野生型小鼠中进行TAC手术和假手术。然后，在手术后2天通过静脉注射将作为对照的AAV(rAAV9-GFP)和表达RalA S28N的AAV(rAAV9-GFP/Flag-RalA S28N)针对小鼠给药(参见图6C)。对照组的AAV感染并没有改善TAC手术引起的心脏功能障碍。在手术后6周与假手术组对比，TAC组中感染AAV对照组的EF和FS两个都显著降低(参见图6D)。重要的是，TAC组中感染表达RalA S28N突变体的AAV的实验组阻止了TAC诱导的EF和FS下降(参见图6D)。这表明RalA S28N的表达有助于维持压力超负荷小鼠心脏的心脏功能。
实施例7：RalGAPα2缺陷导致新生大鼠原代心肌细胞中的SR Ca2+再摄取障碍
在本实施例中，在RalGAPα2的免疫沉淀物中发现SERCA2(参见图8A)。 为了发现RalGAPα2在调节心脏功能中的作用，发明人使用小干扰RNA(siRNA)敲低NRVC中的RalGAPα2，并检查RalGAPα2减少对Ca 2+瞬变的影响。结果发现，Ca 2+瞬变的FDHM和Tau显著增加，而它们的峰值在RalGAPα2减少的NRVC中被压低(参见图8B)。RalGAPα2减少造成的Ca 2+瞬变FDHM和Tau的增加可被RalA S28N逆转(参见图8C)。因此，发明人发现，RalGAPα2复合物与SERCA2的相互作用表明其通过RalA控制SR Ca 2+再摄取来调节Ca 2+胞内平衡。其实验过程已在前文阐释，此处不再赘述。
上述实施例仅供说明本发明之用，而并非是对本发明的限制，有关技术领域的普通技术人员，在不脱离本发明范围的情况下，还可以做出各种变化和变型，因此，所有等同的技术方案也应属于本发明公开的范畴。

Claims (38)

  1. 一种药物组合物在制备预防或治疗心肌病方面药物中的应用，其中所述药物组合物包括：RalGAPα1、RalGAPα2、RalGAPα1或RalGAPα2的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  2. 一种药物组合物在制备预防或治疗心肌病方面药物中的应用，其中所述药物组合物包括：RalGAPα1/β复合物、RalGAPα2/β复合物、RalGAPα1/β复合物或RalGAPα2/β复合物的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  3. 一种药物组合物在制备预防或治疗心肌病方面药物中的应用，其中所述药物组合物包括：RalA、GDP结合形式的RalA、RalA或GDP结合形式的RalA的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  4. 根据权利要求3所述的应用，其中所述药物组合物包括RalA S28N或者GDP结合形式RalA S28N。
  5. 根据权利要求1-4中任一所述的应用，其中所述心肌病由SERCA2介导的钙离子转运异常表征。
  6. 根据权利要求5所述的应用，其中所述心肌病是心脏衰竭。
  7. 根据权利要求6所述的应用，其中所述心肌病是慢性心脏衰竭。
  8. 一种药物组合物在制备正向调节SERCA2a寡聚化药物中的应用，其中所述药物组合物包括：RalGAPα1、RalGAPα2、RalGAPα1或RalGAPα2的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  9. 一种药物组合物在制备正向调节SERCA2a寡聚化药物中的应用，其中所述药物组合物包括：RalGAPα1/β复合物、RalGAPα2/β复合物、RalGAPα1/β复合物或RalGAPα2/β复合物、的剪接变体，截短、片段、取代、添加或删除 突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  10. 一种药物组合物在制备正向调节SERCA2a寡聚化方面药物中的应用，其中所述药物组合物包括：RalA、GDP结合形式的RalA、RalA或GDP结合形式的RalA的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  11. 根据权利要求10所述的应用，其中所述药物组合物包括RalA S28N或者GDP结合形式RalA S28N。
  12. 一种药物组合物在制备正向调节SERCA2活性药物中的应用，其中所述药物组合物包括：RalGAPα1、RalGAPα2、RalGAPα1或RalGAPα2的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  13. 一种药物组合物在制备正向调节SERCA2活性药物中的应用，其中所述药物组合物包括：RalGAPα1/β复合物、RalGAPα2/β复合物、RalGAPα1/β复合物或RalGAPα2/β复合物的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  14. 一种药物组合物在制备正向调节SERCA2活性方面药物中的应用，其中所述药物组合物包括：RalA、GDP结合形式的RalA、RalA或GDP结合形式的RalA的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  15. 根据权利要求14所述的应用，其中所述药物组合物包括RalA S28N或者GDP结合形式RalA S28N。
  16. 一种药物组合物，包括：第一递送载体以及以下活性物质中的一种或多种：(1)RalGAPα1、RalGAPα2、RalGAPα1或RalGAPα2的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白或同系物、同源基因；(2)RalGAPα1/β复合物、RalGAPα2/β复合物、RalGAPα1/β 复合物或RalGAPα2/β复合物的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白或同系物、同源基因；(3)RalA、GDP结合形式的RalA、RalA或GDP结合形式的RalA的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  17. 根据权利要求16所述的药物组合物，其中第一递送载体包括：植入物，脂质体、纳米粒、水凝胶、微球、微囊化递送系统、以及纳米生物医学载体中的一者或多者。
  18. 一种药物组合物，包括：第二递送载体以及编码并能够在心肌细胞中表达以下活性物质中的一种或多种的核酸分子：(1)RalGAPα1、RalGAPα2、RalGAPα1或RalGAPα2的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白或同系物、同源基因；(2)RalGAPα1/β复合物、RalGAPα2/β复合物、RalGAPα1/β复合物或RalGAPα2/β复合物的剪接变体，截短，片段，取代，添加和删除突变，重组突变，基序序列，融合蛋白或同系物、同源基因；(3)RalA、GDP结合形式的RalA、RalA或GDP结合形式的RalA的剪接变体，截短、片段、取代、添加或删除突变体，重组突变体，基序序列，融合蛋白和同系物、同源基因中的一者或多者。
  19. 根据权利要求18所述的药物组合物，其中第二递送载体包括：腺相关病毒、疱疹病毒、牛痘病毒、RNA病毒、胶体分散体系以及纳米生物医学载体中的一者或多者。
  20. 根据权利要求19所述的药物组合物，其包括：使用Ctnt启动子驱动的用于表达RalA S28N突变体的表达盒。
  21. 一种正向调节SERCA2a寡聚化的方法，包括：给予受试者有效量的如权利要求16-20任一所述的药物组合物。
  22. 一种正向调节SERCA2活性的方法，包括：给予受试者有效量的如权利要求16-20任一所述的药物组合物。
  23. 一种预防和治疗受试者心肌病的方法，包括：给予受试者有效量的如权利要求16-20任一所述的药物组合物。
  24. 根据权利要求23所述的方法，其中所述心肌病由SERCA2介导的钙离子转运异常表征。
  25. 根据权利要求24所述的方法，其中所述心肌病是心脏衰竭。
  26. 根据权利要求25所述的方法，其中所述心肌病是慢性心脏衰竭。
  27. 一种鉴定能够影响RalGAPα-RalA信号通路的物质的方法，包括：
    提供RalGAPα-RalA信号通路中蛋白质的多肽或其片段、衍生物、同源物或突变体；
    在允许5’-ATP、5’-GTP和/或5’-GDP与所述多肽结合的条件下，将所述多肽或其片段、衍生物、同源物或突变体与测试物质接触；以及
    确定所述测试物质是否与所述多肽或其片段、衍生物、同源物或突变体结合。
  28. 根据权利要求27所述的方法，其中所述RalGAPα-RalA信号通路中蛋白质包括RalGAPα1、RalGAPα1/β复合物、RalGAPα2、RalGAPα2/β复合物、RalA、GDP结合形式的RalA、以及SERCA2。
  29. 根据权利要求27所述的方法，其中所述RalGAPα-RalA信号通路中蛋白质为GDP结合形式的RalA。
  30. 根据权利要求27所述的方法，进一步包括将已被确定为能够结合所述多肽的物质施用于所述信号通路的任一多肽或其片段、衍生物、同源物或突变体。
  31. 根据权利要求27所述的方法，所述任一多肽或其片段、衍生物、同源物或突变体已经重组产生。
  32. 根据权利要求27所述的方法，进一步包括：确定所述测试物质是否能够替换Ral-GDP信号通路上的所述任一多肽或其片段，而不影响原信号通路的活性。
  33. 一种定量或定性检测受试者SERCA2活性的方法，包括：
    提供与RalGAPα-RalA信号通路中蛋白质相互作用的物质；
    将所述与RalGAPα-RalA信号通路中蛋白质相互作用的物质受试者的细胞，体液或组织接触；以及
    评估受试者RalGAPα-RalA信号通路中蛋白质的含量。
  34. 一种检测受试者RalGAPα-RalA信号通路活性的方法，包括：
    获取受试者的体液或组织；以及
    检测体液或组织的第一细胞的转运Ca 2+能力。
  35. 根据权利要求34所述的方法，进一步包括：
    获取RalGAPα-RalA信号通路相关基因的核酸；
    将获取的核酸表达在第二细胞中；以及
    检测第二细胞的转运Ca 2+能力。
  36. 根据权利要求35所述的方法，所述第二细胞为非洲爪蟾卵母细胞。
  37. 一种诊断受试者心肌病的方法，包括：
    提供与RalGAPα-RalA信号通路中蛋白质相互作用的物质；
    将所述与RalGAPα-RalA信号通路中蛋白质相互作用的物质受试者的细胞，体液或组织接触；以及
    评估受试者RalGAPα-RalA信号通路中蛋白质的含量。
  38. 一种诊断受试者心肌病的方法，包括：通过权利要求33-36任一所述方法检测第一细胞或者第二细胞转运Ca 2+能力。
PCT/CN2022/127868 2021-11-09 2022-10-27 人类受试者心肌病的治疗 WO2023083010A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111318962 2021-11-09
CN202111318962.8 2021-11-09

Publications (1)

Publication Number Publication Date
WO2023083010A1 true WO2023083010A1 (zh) 2023-05-19

Family

ID=81945853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/127868 WO2023083010A1 (zh) 2021-11-09 2022-10-27 人类受试者心肌病的治疗

Country Status (2)

Country Link
CN (1) CN114632147B (zh)
WO (1) WO2023083010A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114632147A (zh) * 2021-11-09 2022-06-17 南京大学 人类受试者心肌病的治疗

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115354043B (zh) * 2022-07-08 2023-12-08 南京大学 一种动物模型

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6605274B1 (en) * 1995-04-11 2003-08-12 The Regents Of The University Of California Method for in vivo regulation of cardiac muscle contractility
WO2007028969A2 (en) * 2005-09-05 2007-03-15 University Of Oslo Anchoring disruption molecules
US20110172144A1 (en) * 2010-01-05 2011-07-14 Celladon Corporation Methods for increasing expression of serca2a in cardiac muscle
CN114632147A (zh) * 2021-11-09 2022-06-17 南京大学 人类受试者心肌病的治疗

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2692052A1 (en) * 2007-06-18 2008-12-24 Celladon Corporation Serca2 therapeutic compositions and methods of use
EP2558113B1 (en) * 2010-04-15 2017-01-11 Celladon Corporation Serca2 therapeutic compositions and methods of use
EP3166609B1 (en) * 2014-07-10 2020-03-11 The Regents of The University of Colorado, A Body Corporate 6-amino-1,3-dimethyl-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrano [2,3-c]pyrazole-5-carbonitrile and related compounds as ral gtpase inhibitors for treating cancer metastasis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6605274B1 (en) * 1995-04-11 2003-08-12 The Regents Of The University Of California Method for in vivo regulation of cardiac muscle contractility
WO2007028969A2 (en) * 2005-09-05 2007-03-15 University Of Oslo Anchoring disruption molecules
US20110172144A1 (en) * 2010-01-05 2011-07-14 Celladon Corporation Methods for increasing expression of serca2a in cardiac muscle
CN114632147A (zh) * 2021-11-09 2022-06-17 南京大学 人类受试者心肌病的治疗

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEN QIAOLI, RONG PING, ZHU SANGSANG, YANG XINYU, OUYANG QIAN, HONG YU, WANG SHUAI: "Targeting RalGAPα1 in skeletal muscle to simultaneously improve postprandial glucose and lipid control", SCIENCE ADVANCES, vol. 5, no. 4, 3 April 2019 (2019-04-03), pages 1 - 15, XP093066935, DOI: 10.1126/sciadv.aav4116 *
KAWAI MIKI, KAWASHIMA SEINOSUKE, SAKODA TSUYOSHI, TOH RYUJI, KIKUCHI AKIRA, YAMAUCHI-TAKIHARA KEIKO, KUNISADA KEITA, YOKOYAMA MITS: "Ral GDP Dissociation Stimulator and Ral GTPase Are Involved in Myocardial Hypertrophy", HYPERTENSION, LIPPINCOTT WILLIAMS & WILKINS, US, vol. 41, no. 4, 1 April 2003 (2003-04-01), US , pages 956 - 962, XP093066944, ISSN: 0194-911X, DOI: 10.1161/01.HYP.0000063884.36641.63 *
LOIRAND GERVAISE, SAUZEAU VINCENT, PACAUD PIERRE: "Small G Proteins in the Cardiovascular System: Physiological and Pathological Aspects", PHYSIOLOGICAL REVIEWS, AMERICAN PHYSIOLOGICAL SOCIETY, US, vol. 93, no. 4, 1 October 2013 (2013-10-01), US , pages 1659 - 1720, XP093066946, ISSN: 0031-9333, DOI: 10.1152/physrev.00021.2012 *
QUAN CHAO, DU QIAN, LI MIN, WANG RUIZHEN, OUYANG QIAN, SU SHU, ZHU SANGSANG, CHEN QIAOLI, SHENG YANG, CHEN LIANG, WANG HONG, CAMPB: "A PKB-SPEG signaling nexus links insulin resistance with diabetic cardiomyopathy by regulating calcium homeostasis", NATURE COMMUNICATIONS, vol. 11, no. 1, XP093066936, DOI: 10.1038/s41467-020-16116-9 *
QUAN CHAO, LI MIN, DU QIAN, CHEN QIAOLI, WANG HONG, CAMPBELL DAVID, FANG LEI, XUE BIN, MACKINTOSH CAROL, GAO XIANG, OUYANG KUNFU, : "SPEG Controls Calcium Reuptake Into the Sarcoplasmic Reticulum Through Regulating SERCA2a by Its Second Kinase-Domain", CIRCULATION RESEARCH, GRUNDE AND STRATTON , BALTIMORE, US, vol. 124, no. 5, 1 March 2019 (2019-03-01), US , pages 712 - 726, XP093066949, ISSN: 0009-7330, DOI: 10.1161/CIRCRESAHA.118.313916 *
ZHU SANGSANG, QUAN CHAO, WANG RUIZHEN, LIANG DERONG, SU SHU, RONG PING, ZHOU KUN, YANG XINYU, CHEN QIAOLI, LI MIN, DU QIAN, ZHANG : "The RalGAPα1–RalA signal module protects cardiac function through regulating calcium homeostasis", NATURE COMMUNICATIONS, vol. 13, no. 1, XP093066934, DOI: 10.1038/s41467-022-31992-z *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114632147A (zh) * 2021-11-09 2022-06-17 南京大学 人类受试者心肌病的治疗
CN114632147B (zh) * 2021-11-09 2024-04-30 南京大学 人类受试者心肌病的治疗

Also Published As

Publication number Publication date
CN114632147B (zh) 2024-04-30
CN114632147A (zh) 2022-06-17

Similar Documents

Publication Publication Date Title
WO2023083010A1 (zh) 人类受试者心肌病的治疗
Liu et al. STAT3 phosphorylation in central leptin resistance
Donato et al. Functions of S100 proteins
Zha et al. ASIC2 subunits target acid-sensing ion channels to the synapse via an association with PSD-95
EP2351573A1 (en) Neuregulin in the treatment of heart diseases
JP2010520209A (ja) Ｎ型カルシウムチャネルを阻害せずに卒中および他の疾患を治療する方法
Li et al. AAV9 gene transfer of cMyBPC N-terminal domains ameliorates cardiomyopathy in cMyBPC-deficient mice
Filomena et al. Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload
US20130196925A1 (en) Compositions and methods useful in enhancement of memory
US20060094101A1 (en) Mk2 interacting proteins
TWI334784B (en) Compositions and methods for suppression of amyloid plaque formation associated with neurodegenerative disorders
EP2434288B1 (en) Means and methods for diagnosing and/or treating a subject at risk of developing heart failure
WO2021105137A1 (en) Trem-1 inhibitors for the treatment of vaso-occlusions and tissue injuries in patients suffering from sickle cell disease
Wu et al. Age-related changes in P-glycoprotein expression in senescence-accelerated mouse
Wu et al. Lysophosphatidic acid contributes to myocardial ischemia/reperfusion injury by activating TRPV1 in spinal cord
JP6931218B2 (ja) 細胞膜の損傷修復促進剤
US20020197737A1 (en) Novel methods for testing inhibitors of paired helical filaments and uses for treatment of alzheimer&#39;s disease
EP1296138A1 (en) Use of disease-associated gene
WO2022170184A1 (en) Lipocalin 10 as a therapeutic agent for inflammation-induced organ dysfunction
Zhou et al. The Mechanism of SIRT3 Regulating SRV2-Mediated Mitochondrial Fission of Fibroblasts to Inhibit Myocardial Fibrosis after Acute Myocardial Infarction
US20090227501A1 (en) Agents for preventing and/or treating upper digestive tract disorders
JP2006525244A (ja) 糖尿病の治療標的としてのインスリン誘導性遺伝子
EP1600165A1 (en) Medicinal use of mip-3alpha inhibitor and method of screening brain/nerve cell protective agent
Grämer Development of a GRK2-inhibitory peptide
Handschin et al. regulates the neuromuscular junction program and α PGC-1

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22891806

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE