Use of inhibitors of super-elongation complexes in heart failure therapy

Abstract

The present disclosure relates to the field of biomedicine, and particularly relates to the application of super elongation complex inhibitor in heart failure treatment. The present disclosure first discovers the important role of super elongation complex inhibitor in heart failure treatment, so that a new drug for treating heart failure can be found.

Description

Technical Field

[0001] This disclosure generally relates to the field of biomedical technology, and more specifically, to the application of inhibitors of super-extended complexes in the treatment of heart failure. Background Technology

[0002] The Super Elongation Complex (SEC) is a multi-protein complex that promotes the release and elongation of RNA polymerase II (RNAPolII) transcriptional arrest. The main components of the SEC include AFF4, ELL2, EAF1 / EAF2, and P-TEFb. AFF4 acts as a scaffold, binding to ELL2 and simultaneously binding to the P-TEFb subunit CCTN1 to recruit P-TEFb. Subsequently, the P-TEFb subunit CDK9 phosphorylates RNAPolII, promoting the release and arrest of RNAPolII, thereby accelerating transcriptional elongation and regulating the rapid and coordinated transcriptional activation of stress-related and developmental genes.

[0003] The SEC complex plays a crucial role in development, HIV gene transcription, and rapid cellular responses to the environment. SEC is involved in the development of various cancers, such as myeloid leukemia and lymphocytic leukemia with high Myc expression. SEC is a co-regulator of the human immunodeficiency virus (HIV) Tat protein, regulating viral replication. SEC also participates in the rapid expression of heat stress antiproteins, exhibiting different characteristics and target genes compared to transcription mediated by the BRD4 / P-TEFb complex. However, the expression and function of SEC in cardiovascular diseases such as heart failure remain unexplored. Summary of the Invention

[0004] This invention covers the following technical solutions:

[0005] (1) The use of inhibitors of super-extended complex (SEC) in the preparation of drugs for the treatment of heart failure.

[0006] (2) According to the application described in (1), the inhibitor targets one or more of the SEC subunits.

[0007] (3) According to the application described in (2), the SEC subunit includes one or more of the following: EAF1, EAF2, ELL1, ELL2, ELL3, AFF1, AFF2, AFF3, AFF4, AF9, ENL, and P-TEFb.

[0008] (4) According to the application described in (3), the inhibitor targets SEC subunits including P-TEFb and targets CDK9 and / or cyclin T1 subunits therein.

[0009] (5) According to the application described in (3), the SEC subunit includes at least one or more of ELL2, AFF4, and P-TEFb.

[0010] (6) According to any one of (2) to (5), the inhibitor comprises at least one of antisense oligonucleotide, dsRNA, siRNA, shRNA, miRNA, RNAi and ribozyme, which is designed to reduce or block the expression of the SEC subunit.

[0011] (7) According to any one of (2) to (5), the inhibitor comprises at least one of an antibody or an antigen-binding fragment thereof, a small molecule compound, and an aptamer, which is designed to reduce or counteract the activity of the SEC subunit and / or block the assembly of the SEC.

[0012] (8) According to the application described in (1), the inhibitor blocks the interaction between AFF4 and P-TEFb subunits (especially cyclin T1).

[0013] (9) According to the application described in (8), the inhibitor includes KL-1 and / or KL-2.

[0014] (10) The application according to any one of claims (1) to (9), wherein the inhibitor knocks down or removes the gene of the SEC subunit or the gene that regulates the expression of the SEC subunit by ZFN technology, TALEN technology or CRISPR technology.

[0015] (11) According to the application described in (10), the inhibitor comprises gRNA and CRISPR enzyme, or a vector capable of expressing them.

[0016] (12) The inhibitor is used alone or in combination with other drugs according to any one of (1) to (11).

[0017] (13) According to the application described in (12), the other drugs include at least one of the following: diuretics, renin-angiotensin system inhibitors, vasodilators, aldosterone antagonists, β-adrenergic receptor blockers, MRA, sodium-glucose cotransporter 2 inhibitors, sinoatrial node pacemaker current inhibitors, rehmannia-like drugs, guanylate cyclase stimulants, and myosin agonists.

[0018] (14) The inhibitor is used in any one of (1) to (13) to form a pharmaceutical composition with a pharmaceutically acceptable excipient.

[0019] (15) According to the application described in (14), the pharmaceutical composition is selected from one or more of the following: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule and granule.

[0020] (16) According to any one of (1) to (15), the heart failure is heart failure with preserved ejection fraction or heart failure with decreased ejection fraction.

[0021] (17) According to any one of (1) to (16), the efficacy of the drug includes at least one of inhibiting left ventricular dilation, inhibiting cardiac fibrosis, inhibiting cardiomyocyte hypertrophy, and inhibiting cardiac cell apoptosis.

[0022] (18) In any of the applications described in (1) to (17), the patient of the drug is a mammal.

[0023] (19) According to the application described in (18), the patient with the drug is a primate.

[0024] (20) According to the application described in (19), the patient of the drug is a human being.

[0025] Beneficial effects:

[0026] This invention is the first to discover the important role of inhibitors of super-extended complexes in the treatment of heart failure, thereby enabling the search for new drugs to treat heart failure. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 Aortic arch constriction (TAC) induces heart failure and upregulates the expression of ELL2, a component of the transcriptional super-elongation complex, in the heart of a heart failure patient.

[0029] Figure 2 Overexpression of ELL2 in cardiomyocytes does not affect cardiac function under normal physiological conditions, but it can promote the pathological development of heart failure.

[0030] Figure 3 Conditional knockout of ELL2 in cardiomyocytes does not affect cardiac function under normal physiological conditions, but it can inhibit the pathological development of heart failure.

[0031] Figure 4 Conditional knockout of ELL2 in cardiomyocytes does not affect the heart structure under normal physiological conditions, but it can inhibit structural lesions in heart failure.

[0032] Figure 5ELL2 expression is upregulated in AngII-stimulated cardiomyocytes, and knocking out ELL2 can inhibit the AngII-induced cardiomyocyte hypertrophy phenotype.

[0033] Figure 6 RNA-Seq showed that conditional knockout of ELL2 in cardiomyocytes downregulated the expression of genes related to extracellular matrix and myostructural development in heart failure, while overexpression of ELL2 in cardiomyocytes upregulated the expression of genes related to extracellular matrix and myostructural development in heart failure.

[0034] Figure 7 GSEA analysis showed that conditional knockout of ELL2 in cardiomyocytes downregulated the expression of genes related to fibrosis, cardiac hypertrophy, and inflammation in heart failure, while overexpression of ELL2 in cardiomyocytes upregulated the expression of these genes.

[0035] Figure 8 qPCR experiments showed that conditional knockout of ELL2 in cardiomyocytes downregulated the expression of fibrosis-related genes in the heart of heart failure, while overexpression of ELL2 in cardiomyocytes upregulated the expression of these genes.

[0036] Figure 9 Immunoblotting experiments showed that RNA polymerase II S2 phosphorylation and ELL2 expression were increased in the heart of heart failure, while conditional knockout of ELL2 in cardiomyocytes downregulated RNA polymerase II S2 phosphorylation and ELL2 expression in the heart of heart failure, while the expression of other SEC complex components such as AFF4 and BRD4 was not affected.

[0037] Figure 10 Mice treated with KL-1 on the second day after heart failure was induced by TAC surgery showed that the assembly of the ELL2 / AFF4 SEC complex was inhibited.

[0038] Figure 11 Treatment with KL-1 on the second day after inducing heart failure in mice via TAC surgery improved cardiac function.

[0039] Figure 12 In mice, treatment with KL-1 on the second day after inducing heart failure via TAC surgery inhibited heart failure-related cardiac dilatation, fibrosis, and cardiomyocyte hypertrophy.

[0040] Figure 13 qPCR experiments showed that KL-1 treatment on the second day after heart failure was induced by TAC surgery in mice could inhibit heart failure-related cardiac dilation and fibrosis.

[0041] Figure 14 Four weeks after inducing heart failure in mice via TAC surgery, treatment with KL-1 improved heart failure-related decline in cardiac function.

[0042] Figure 15 Four weeks after inducing heart failure in mice via TAC surgery, treatment with KL-1 reversed cardiac hypertrophy caused by heart failure. Detailed Implementation

[0043] Reference will now be made to embodiments of this disclosure in detail, with one or more examples described below. Each example is provided for explanation and not for limitation of this disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to this disclosure without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0044] Unless otherwise stated, all terms used in the disclosure of this disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further guidance is provided below for a better understanding of the teachings of this disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure.

[0045] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0046] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this disclosure, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0047] The terms “containing,” “including,” and “comprise” as used in this disclosure are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0048] The range of values ​​represented by endpoints in this disclosure includes all values ​​and fractions contained within that range, as well as the endpoints referenced.

[0049] When this document uses the term "about" to refer to a value or parameter, it includes (and describes) an implementation of the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0050] The concentration values ​​mentioned in this disclosure include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may allow fluctuations within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are allowed. For example, 100mM may allow fluctuations within the ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations within ±10% are allowed.

[0051] As used herein, unless otherwise indicated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0052] The phrase “for example” should be interpreted as “for example, including”. Furthermore, the use of any and all exemplary language, including but not limited to “for example”, is merely for the purpose of better illustrating the invention and does not limit the scope of the invention unless otherwise required.

[0053] In this disclosure, the terms "multiple" or "various" unless otherwise specified refer to a quantity of 2 or more.

[0054] In this disclosure, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0055] In this disclosure, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments and should be understood not to limit the scope of protection of this disclosure. In this disclosure, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" options appear in a technical solution, unless otherwise specified and without contradictions or mutual constraints, each "optional" option is independent.

[0056] In this disclosure, the term "Super Elongation Complex" (SEC) is used interchangeably with "Super Transcriptional Elongation Complex," "Super Elongation Complex," and "Super Elongation Complex." "SEC" refers to the assembled complex, and "SEC subunit" refers to the domain, structural fragment, or secondary structural unit that makes up the SEC; it can also be replaced by "SEC component." Subunits may include one or more of EAF1, EAF2, ELL1, ELL2, ELL3, AFF1, AFF2, AFF3, AFF4, AF9, ENL, and P-TEFb (CDK9 / cyclin T1).

[0057] In this disclosure, the term "inhibitor of the super-elongation complex" refers to a substance that reduces the bioavailability of SEC. Specifically, an inhibitor refers to a substance having at least one of the following functions: knocking down or eliminating the gene of the SEC subunit; reducing or blocking the expression of the SEC subunit; reducing or blocking the translation of the SEC subunit; reducing or counteracting the activity of the SEC / SEC subunit (e.g., blocking or reducing its binding to downstream effector molecules such as pol II transcription genes); hindering or halting the assembly of SEC; or inducing the degradation of the SEC / SEC subunit. The inhibitors used in this disclosure include expression inhibitors and function inhibitors. As known to those skilled in the art, inhibitors can inhibit proteins at the genomic level, transcriptional level, translational level, post-translational modification level, etc., as long as they reduce the amount of functional protein. Examples of post-translational modifications include the maturation of gene products or proteins, and post-translational modifications (e.g., glycosylation, disulfide bonding, tetradecylation, protease cleavage, binding to other proteins, ubiquitous proteinization, etc.). Protein processing, transport, and release can also be modified, for example, by placing them in storage organelles before release, or by binding to other proteins that affect release.

[0058] As used herein, the terms “assembly,” “bonding,” “interaction,” “occupation,” and “occupation” refer to covalent interactions, non-covalent interactions, and spatial interactions, or combinations thereof. A covalent interaction is a chemical bond between two atoms or groups formed by sharing a single pair of electrons (single bond), two pairs of electrons (double bond), or three pairs of electrons (triple bond). Covalent interactions are also referred to in this field as electron pair interactions or electron pair bonds. Non-covalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (through short-range non-covalent forces), hydrophobic interactions, ionic bonds, etc. For a commentary on non-covalent interactions, see Alberts et al., Molecular Biology of the Cell, 3D edition, Garland Publishing, 1994. Spatial interactions are generally understood to include those in which the structure of a compound allows it to occupy a site through its three-dimensional structure, rather than any attraction between the compound and the site.

[0059] As used in this article, the term "heart failure" (HF), also known as cardiac failure, refers to a clinical syndrome characterized by the inability of the heart to pump enough blood to meet the needs of the tissues. This is caused by initial myocardial damage (myocardial infarction, excessive hemodynamic load, inflammation, etc.) from any cause, resulting in changes in myocardial structure and function, leading to cardiac systolic and diastolic dysfunction. It is usually accompanied by elevated serum natriuretic peptide levels and / or objective evidence of cardiogenic pulmonary or systemic congestion on imaging examinations at rest or during exercise, or elevated ventricular filling pressure on hemodynamic examinations. Based on the speed of its progression, heart failure can be classified in this paper as acute heart failure and chronic heart failure (congestive heart failure); based on anatomical location, it can be classified as left heart failure, right heart failure, and biventricular heart failure; based on physiological dysfunction, it can be classified as systolic heart failure and diastolic heart failure; or heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF); based on disease progression, it can be classified as stage A (heart failure risk period), stage B (pre-heart failure), stage C (symptomatic heart failure), and stage D (late heart failure).

[0060] In this specification, “treatment” means (1) delaying the onset of heart failure; (2) slowing or stopping the progression, worsening or exacerbation of heart failure symptoms; (3) bringing relief of heart failure symptoms; or (4) a method or procedure for curing heart failure. As a preventative measure, treatment may be performed before the onset of the disease or condition, or treatment may be performed after the onset of the disease.

[0061] As used in this article, the term "specific binding" refers to a higher affinity of a binding molecule for a target molecule compared to its affinity for non-target molecules. A binding molecule that specifically binds to a target molecule essentially does not recognize or bind to non-target molecules; for example, an antibody "specifically binds" and / or "specifically recognizes" another molecule, meaning that this interaction depends on the presence of binding specificity of the molecular structure, such as an antigenic epitope.

[0062] As used herein, the term “treatment” and its variations or “improvement” refers to therapeutic treatment in which the aim is to reverse, reduce, improve, suppress, slow, or stop the progression or severity of a disease (e.g., heart failure), its associated symptoms, and / or conditions. The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a disease (e.g., heart failure).

[0063] As used in this invention, "therapeuticly effective" and "effective dose" refer to the substance or amount that causes the desired biological activity or effect.

[0064] Unless otherwise stated, the terms "subject" or "patient" are used interchangeably to refer to mammals, such as primates (and further, human patients), and laboratory animals, such as rabbits, rats, and mice, as well as other animals. Therefore, as used herein, the terms "subject" or "patient" refer to any mammalian patient or subject to whom the compounds of the present invention may be administered. In exemplary embodiments of the present invention, in order to identify patients to be treated according to the methods of the present invention, acceptable screening methods are employed to determine risk factors associated with the target or suspected disease or condition, or to determine the subject's existing disease or condition. These screening methods include, for example, routine examinations to determine risk factors that may be associated with the target or suspected disease or condition. These and other conventional methods allow clinicians to select patients requiring treatment using the methods and compounds of the present invention.

[0065] Throughout this specification, when a composition is described as having, including, or comprising specific components, or when a method is described as having, including, or comprising specific process steps, it is contemplated that the compositions of the present invention also substantially consist of or comprise the following: the methods of the present invention also substantially consist of or comprise the listed processing steps.

[0066] All documents mentioned in this disclosure are cited herein as if they were cited individually. Unless they conflict with the inventive purpose and / or technical solution of this disclosure, all cited documents are cited in their entirety and for all purposes. When citing documents in this disclosure, the definitions of relevant technical features, terms, nouns, phrases, etc., are also cited. When citing documents in this disclosure, examples and preferred embodiments of the relevant technical features may also be incorporated into this disclosure as reference, but only to the extent that they enable the implementation of this disclosure. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or modifications shall be made adaptively based on the description in this disclosure.

[0067] This invention relates to the use of inhibitors of super-extended complexes (SECs) in the preparation of medicaments for the treatment of heart failure.

[0068] In some implementations, the inhibitor targets one or more of the SEC subunits.

[0069] In some implementations, the SEC subunit includes one or more of the following: EAF1, EAF2, ELL1, ELL2, ELL3, AFF1, AFF2, AFF3, AFF4, AF9, ENL, and P-TEFb.

[0070] In some implementations, the SEC subunit includes at least ELL2.

[0071] In some implementations, the SEC subunit includes at least AFF4.

[0072] In some implementations, the SEC subunit includes at least P-TEFb.

[0073] In some implementations, the SEC subunit includes at least the cyclin T1 subunit.

[0074] In some implementations, the SEC subunit includes at least ELL2 and / or AFF4.

[0075] In some implementations, the SEC subunit includes at least ELL2 and / or P-TEFb.

[0076] In some implementations, the SEC subunit includes at least AFF4 and / or P-TEFb.

[0077] In some implementations, the SEC subunit includes at least one or more of ELL2, AFF4, and P-TEFb.

[0078] In some implementations, the SEC subunit includes at least one or more of ELL2, AFF1, AFF2, AFF3, and AFF4.

[0079] In some embodiments, the inhibitor targets SEC subunits including P-TEFb, and specifically targets the CDK9 and / or cyclin T1 subunit (CCNT1). In this invention, P-TEFb can be in an activated or inactivated state.

[0080] In some implementations, the inhibitor specifically targets the SEC subunit / the SEC.

[0081] SEC activity can be evaluated using methods known in the art and methods disclosed herein, including those disclosed in the embodiments provided herein. In some embodiments, the inhibitor disrupts and / or reduces the biological activity of SEC (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) relative to a control. In other embodiments, IC 50 Numerical tables can determine the IC50 values ​​of inhibitors that disrupt SEC and / or reduce SEC biological activity, preferably the IC50 values ​​of the compounds. 50 Values ​​less than approximately 10 mM, 5 pm, or 1 pm, 0.5 pm, 0.1 pm, 0.05 pm, 0.01 pm, 0.005 pm, or 0.001 pm.

[0082] In some embodiments, the inhibitor comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi, and ribozymes, which is designed to reduce or block the expression of the SEC subunit.

[0083] siRNA refers to double-stranded RNA that inhibits the expression of target nucleic acids; it is also known as "RNAi reagent," "short-strand interfering RNA," "short-strand interfering nucleic acid," and "siRNA." The nucleic acid molecule can inhibit or downregulate gene expression or viral replication through sequence-specific RNA interference (RNAi) or gene silencing. It can consist of only RNA or be a fusion of DNA and RNA. siRNA can be prepared using conventional methods based on information obtained by searching for target nucleic acids and the NCBI database.

[0084] Ribozymes are RNA molecules that specifically cleave other single-stranded RNA molecules through a mechanism similar to DNA restriction endonucleases. Ribozymes that recognize and cleave specific base sequences in the single strand of RNA can be manufactured by appropriately modifying the nucleic acid sequence of the RNA using known techniques (Science, 239, p. 1412-1416, 1988).

[0085] In one embodiment of this disclosure, substances that specifically bind to SEC subunit proteins can be cited as inhibitors of SEC. Examples of substances specifically binding to SEC subunit proteins include substances selected from the group consisting of antibodies, antibody fragments, small molecule compounds, and aptamers. These substances can be designed to reduce or counteract the activity of said SEC subunits and / or inhibit SEC assembly. When designed to inhibit SEC assembly, interactions between any two or more SEC subunits can be inhibited. The binding site can be the assembly junction. For example, the SEC components ENL and AFF4 can act as a scaffold to dynamically isolate and concentrate the kinase module P-TEFb of SEC from the inactive HEXIM1-P-TEFb complex via LLPS, thereby promoting the rapid assembly of SEC. Inhibitors can bind to surface epitopes of AFF4, for example, to create steric hindrance and prevent its binding to other subunits such as p-TEF subunits (especially cyclin T1). Inhibitors can also bind to surface epitopes of both AFF4 and p-TEF subunits (especially cyclin T1) to similarly prevent assembly. For example, compounds with this function are described in Liang K, Smith ER, Aoi Y, et al. Targeting processive transcription elongation via SEC disruption for MYC-induced cancer therapy[J]. Cell, 2018, 175(3):766-779.e17. The relevant part is incorporated herein by reference.

[0086] Antibodies include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies. Examples of antibodies include full-length antibodies, single-chain molecules, bifunctional molecules, scFv, exody, single-domain antibodies (VHH), chimeric antibodies, and immunoadhesion. "Antibody fragment" includes fragments of Fv, Fv′, Fab, Fab′, and F(ab′)2. Where this disclosure applies, "antibody" means a polypeptide containing one or more regions that bind to epitopes of a target antigen. Antibodies against OASL can be prepared by any method known in the art. For example, monoclonal antibodies may be generated by immunizing mice with recombinant OASL or one or more synthetic peptides (including epitopes considered highly antigenic). Alternatively, polyclonal antibodies may be generated by immunizing rabbits with the aforementioned antigen. Once generated using hybridoma technology, the monoclonal antibody is preferably humanized through the exchange of mouse and human backbones and constant regions.

[0087] Antibody fragments can be obtained by treating antibodies with proteases such as papain and pepsin (Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science (1985) 229: 81). Antibodies can also be generated through genetic recombination based on the amino acid sequence of antibody fragments.

[0088] Low molecular weight antibodies with modified structures containing “antibody fragments” can be constructed using antibody fragments obtained through enzymatic treatment or gene recombination. Alternatively, a gene encoding the entire low molecular weight antibody can be constructed and introduced into an expression vector, which is then expressed in a suitable host cell (e.g., Co et al., J. Immunol.). (1994) 152: 2968-76; Better and Horvitz, Method Enzymes. (1989) 178: 476-96; pluckthun and Skerra, Method Enzymes. (1989) 178: 497-515; Lamoy, Method Enzymes. (1986) 121: 652-63; Rouseaux et al., Methods Enzymol. (1986) 121: 663-9; Byrd and Walker, Trends Biotechnology. (1991) 9: see 132-7.

[0089] In this disclosure, an aptamer is a nucleic acid or peptide molecule that binds to a specific target molecule. Aptamers are typically generated by selecting them from a large pool of random arrays, but naturally occurring aptamers also exist on riboswitches. These aptamers can be used as polymeric pharmaceuticals for basic research and clinical purposes. Aptamers can be combined with self-cleaving ribozymes in the presence of the target molecule. More specifically, aptamers can be classified as nucleic acid aptamers, such as DNA or RNA aptamers, or as peptide aptamers. The former typically consists of oligonucleotide chains (usually shorter), while the latter preferably consists of short, variable peptide domains attached to both ends of a protein scaffold. In principle, nucleic acid aptamers bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and even the organism itself. In vitro selection or equivalent SELEX (systematic evolution of exponentially enriched ligands) is a manipulation of nucleic acid species through repeated rounds of enrichment (enrichment). Peptide aptamers are typically designed to interfere with interactions between other proteins in the cell. They consist of variable peptide loops attached to both ends of a protein scaffold. This dual-structure constraint increases the binding affinity of the peptide aptamer to a level large and equal to that of the antibody (nanomolar range). The variable peptide ring typically contains 10-20 amino acids, and the scaffold can be any protein with good solubility properties. Currently, bacterial thioredoxin A is the most commonly used scaffold protein, with the wild-type protein containing the -Cys-Gly-Pro-Cys-loop. In the variable peptide ring embedding at the redox active site, two cysteine ​​residues in the side chain can form disulfide crosslinks. Peptide aptamers can be selected using different systems, but the yeast-to-hybrid system is currently the most widely used.

[0090] In some embodiments, the inhibitor knocks down or removes genes of the SEC subunit or genes that regulate the expression of the SEC subunit.

[0091] Knockdown or removal can be achieved using any known existing technology, such as zinc finger ribonuclease (ZFN) technology, TALEN (transcription activator-like effector nucleases) technology, or CRISPR technology.

[0092] In some embodiments, the inhibitor comprises gRNA and a CRISPR enzyme, or a vector capable of expressing them. In some embodiments, the antagonist is a CRISPR-Cas gene editing composition / combination product. In some embodiments, it comprises gRNA and a CRISPR enzyme (e.g., Cas9) or a vector capable of expressing them. There are two common gene knockout strategies: one is single sgRNA-mediated gene knockout, which uses the CRISPR-Cas9 system to cause frameshift mutations in the InDels generated in the exons of the coding gene, thereby achieving gene knockout; therefore, this gene knockout strategy is limited to the knockout of coding genes. The other is two sgRNA-mediated gene knockout, where two sgRNAs act on both sides of the target fragment to delete the target fragment sequence, thereby achieving the knockout of the target gene.

[0093] In some embodiments, the inhibitor comprises a small molecule compound that is designed to induce the degradation of SEC.

[0094] In some embodiments, the inhibitor includes KL-1 and / or KL-2.

[0095] For details of KL-1 and KL-2, see WO2019183373A1 and its family of patents, such as US11453640B2, the relevant portions of which are incorporated herein by reference.

[0096] For small molecule compounds such as KL-1 and / or KL-2, their administration forms are typically pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that is substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those prepared by reacting the compounds disclosed herein with pharmaceutically acceptable inorganic or organic acids or bases. These salts are referred to as acid addition salts and base addition salts. Those skilled in the art will recognize that most or all of the compounds disclosed herein are capable of forming salts, and that the salt form of the drug is typically used, usually because they are easier to crystallize and purify than free acids or bases.

[0097] Acids commonly used to form acid addition salts can include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., as well as organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Suitable pharmaceutically acceptable examples of salts may include sulfates, pyrosulfates, bisulfates, sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, bromides, iodides, acetates, propionates, decanoates, caprylates, octanoates, acrylates, formates, hydrochlorides, dihydrochlorides, isobutyrates, hexanoates, heptanoates, propionates, oxalates, malonates, succinates, octanoates, sebacic acid esters, fumarates, maleic acid-, butyn-1,4-diesters, hexyn-1,6-diesters, benzoates, chlorobenzoates, methyl benzoate, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenyl acetate, phenyl propionate, phenyl butyrate, citrates, lactates, hydroxybutyrates, glycolates, tartrates, methanesulfonates, propionate sulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, etc.

[0098] Base addition salts include those derived from inorganic bases, such as ammonium or alkali metal or alkaline earth metal hydroxides, carbonates, bicarbonates, etc. Bases used to prepare these salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, etc.

[0099] Furthermore, the methods disclosed herein can be implemented using solvates of the compounds disclosed herein. Solvates may include ethanol solvates, hydrates, etc.

[0100] The compounds used in the methods disclosed herein can be formulated into pharmaceutical compositions comprising one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers may include lactose monohydrate, anhydrous lactose, and various starches; examples of binders are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose such as AVICELPH101 and AVICELPH102, microcrystalline cellulose, and silicified microcrystalline cellulose (PROSOLVSMCC). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silica such as Aerosil 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweeteners such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame potassium. Examples of flavoring agents are MAGNASWEET (a trademark of MAFCO), bubble gum flavoring, and fruit flavorings, etc. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of p-hydroxybenzoic acid such as butylparaben, alcohols such as ethanol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0101] In some implementations, the inhibitor is used alone or in combination with other drugs.

[0102] In some embodiments, the other drugs include at least one of the following: diuretics (e.g., loop diuretics, such as furosemide, torasemide, bumetanide, tolvaptan), renin-angiotensin system (RAS) inhibitors (e.g., ACEIs, ARBs, and ARNIs), vasodilators (e.g., hydralazine, isosorbide dinitrate, nitroglycerin patches or sprays), aldosterone antagonists, β-adrenergic receptor blockers (e.g., bisoprolol or metoprolol succinate extended-release tablets or carvedilol), MRAs (represented by spironolactone or eplerenone), sodium-glucose cotransporter 2 inhibitors (e.g., dapagliflozin), sinoatrial node pacemaker current inhibitors (e.g., ivabradine), rehmannia-like drugs (e.g., digoxin), guanylate cyclase stimulants (e.g., veliciguat), and cardiac myosin agonists (e.g., Omecamtiv mercabil).

[0103] In some embodiments, the inhibitor is combined with pharmaceutically acceptable excipients to form a pharmaceutical composition. The pharmaceutical composition can be formulated using methods known to those skilled in the art. For example, it can be administered parenterally as an injection of a sterile solution or suspension of water or a pharmaceutically acceptable solution other than water. For example, a suitable combination of pharmacologically acceptable carriers or mediators, specifically sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, mediators, preservatives, binders, etc. It is considered to be formulated by mixing in a unit dosage form required for generally accepted pharmaceutical administration. The amount of the active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0104] Sterile compositions for injection can be formulated using excipients such as distilled water for injection, following conventional formulation methods.

[0105] Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, lactose, glucose, and other supplements (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate dissolving aids can be used in combination, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80(TM), HCO-50, etc.).

[0106] As an oily liquid, sesame oil and soybean oil are examples, and benzyl benzoate and / or benzyl alcohol can be used in combination as a solubilizing agent. The composition can be mixed with buffers (e.g., phosphate buffer solutions and sodium acetate buffer solutions), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is typically packaged in appropriate ampoules.

[0107] The pharmaceutical composition is preferably administered via parenteral administration. For example, in some embodiments, the pharmaceutical composition is selected from one or more of the following forms: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule, and granule. For example, it can be administered systemically or locally, such as by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0108] Suitable pharmaceutically acceptable carriers are well known in the art. Descriptions of some of these pharmaceutically acceptable carriers can be found in the Handbook of Pharmaceutical Excipients published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain. Furthermore, techniques and compositions for preparing useful dosage forms using the present invention are described in one or more of the following references: Pharmacopoeia of the People's Republic of China 2020 Edition; United States Pharmacopeia (USP#24NF19) (Hardcover Text w / 3 Supplements) (USP 2002); Remington: The Science and Practice of Pharmacy (21st edition, formerly Remington's Pharmaceutical Sciences), Mack Publishing Co., 2005); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992), etc., relevant portions of which are incorporated herein by reference.

[0109] In some implementations, the heart failure is heart failure with preserved ejection fraction or heart failure with decreased ejection fraction.

[0110] In some embodiments, the efficacy of the drug includes at least one of inhibiting left ventricular dilation, inhibiting cardiac fibrosis, inhibiting cardiomyocyte hypertrophy, and inhibiting cardiac cell apoptosis.

[0111] According to the above-described application of the present invention, a drug for treating heart failure can be identified, which contains an inhibitor of the super-extended complex (SEC) as the main active ingredient.

[0112] A pharmaceutical composition for treating heart failure is also provided, comprising an inhibitor of SEC and other drugs, more specifically other drugs for treating heart failure.

[0113] A pharmaceutical composition for treating heart failure is also provided, comprising an inhibitor of SEC and pharmaceutically acceptable excipients.

[0114] Also provided is a pharmaceutical composition for treating heart failure, comprising an inhibitor of SEC and other drugs (more specifically, other drugs for treating heart failure), and pharmaceutically acceptable excipients.

[0115] Furthermore, the descriptions in the above applications also apply to the drugs / drug compositions for treating heart failure as described above.

[0116] The present invention also relates to a method of treating a patient with heart failure, comprising administering to the patient a pharmaceutical composition wherein the active ingredient of the pharmaceutical composition contains an effective amount of at least one inhibitor of SEC.

[0117] The method of administration can be appropriately selected based on the patient's age and symptoms. The dosage of the active ingredient can be set in, for example, the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, the dosage can be 0.001-100000 mg (e.g., about 0.01, 0.1, 1, 10, 15, 25, 50, 100, 125, 150, 175, 200, 250, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 mg or even higher) per patient, but this disclosure is not necessarily limited to these values. The frequency of administration can be once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times weekly to treat the subject's disease or condition. The dosage and method of administration vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can consider these factors to determine an appropriate dosage and method of administration.

[0118] Those skilled in the art will recognize that in vivo and in vitro assays using appropriate, known and generally accepted cell and / or animal models can predict the ability of a SEC inhibitor to treat or prevent a given condition. Those skilled in the art will further recognize that human clinical trials can be conducted using clinically well-known methods, including first-in-human trials, dose-range trials, and efficacy trials in healthy subjects and / or subjects with a given disease.

[0119] In some embodiments, the pharmaceutical composition is used in combination with other drugs (preferably drugs for treating heart failure other than SEC inhibitors) to treat patients with heart failure. The pharmaceutical composition and other drugs may be administered together (e.g., to form a new pharmaceutical composition) or separately at intervals.

[0120] In some embodiments, the pharmaceutical composition is used in combination with at least one other method of treating heart failure besides drug therapy to treat patients with heart failure. Methods of treating heart failure include: exercise training (e.g., supervised exercise training for 3–6 months), implantable cardiac electronic devices (such as implantable cardioverter-defibrillators (ICDs), cardiac resynchronization therapy (CRT), and cardiac contractility modulators (CCMs)), lifestyle management (including avoiding excessive water intake, excessive intravenous fluids, limiting sodium intake, adopting a low-fat or diabetic diet, and managing triggers such as alcoholism and infection), heart transplantation, coronary artery bypass surgery, and ventricular assist devices (VADs).

[0121] Furthermore, the description in the above application / pharmaceutical composition also applies to the methods described above.

[0122] The embodiments of this disclosure will now be described in detail with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. For experimental methods in the following embodiments where specific conditions are not specified, reference should be made to the guidelines given in this disclosure, or to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to the conditions recommended by the manufacturer.

[0123] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0124] Example 1: Upregulation of ELL2 expression in the heart of a heart failure patient

[0125] Adult male C57BL / 6J mice (8 weeks old) were randomly divided into two groups. The experimental group underwent aortic arch coarctation (TAC) to induce heart failure. Four weeks after TAC, qPCR was used to detect the expression of key components of the SEC complex in the heart. It was found that ELL2 expression was upregulated in the TAC experimental group, while ELL1 and ELL3 expression were unaffected. Figure 1 Immunoblotting experiments also demonstrated that ELL2 expression was upregulated in the hearts of mice with TAC-induced heart failure, see [reference needed]. Figure 1 B.

[0126] Example 2: Overexpression of ELL2 in the heart of a heart in patients with heart failure can promote the progression of heart failure.

[0127] Wild-type (WT) C57BL / 6J mice were randomly divided into two groups at neonatal period (3 days after birth, P3). The experimental group was subcutaneously injected with cTnT-ELL2 adeno-associated virus 9, while the control group was injected with cTnT-tdTomato adeno-associated virus 9, inducing cardiomyocyte-specific overexpression of ELL2 or tdTomato. Both groups of mice underwent transcatheter arthroplasty (TAC) to induce heart failure at 8 weeks of age, while the control group underwent sham surgery (n=6). Four weeks after TAC, echocardiography revealed no significant difference in cardiac function between ELL2 and tdTomato overexpression in the sham-operated group, indicating that ELL2 overexpression does not affect cardiac function under physiological conditions. However, in the TAC-operated group, compared to mice overexpressing tdTomato, mice overexpressing ELL2 showed a more severe reduction in contractile function (ELL2...). OE vs tdTomatoOE,FS:% vs%,P<0.0001) and ventricular dilation phenotype (ELL2) OE vs tdTomatoOE,IVS+LVPWd:mm vs mm,P<0.05), see Figure 2 A.

[0128] Anatomical findings of the heart four weeks after TAC surgery also showed that hearts overexpressing ELL2 were significantly enlarged. Figure 2 HE staining also showed significant left ventricular dilation in hearts overexpressing ELL2, see [link to article]. Figure 2 C. WGA staining also showed that cardiomyocytes overexpressing ELL2 had a significantly larger cross-sectional area. See Figure 2 Medium DE (n=4).

[0129] Example 3: Knocking out ELL2 in the heart of a heart in patients with heart failure can inhibit the progression of heart failure.

[0130] To further confirm the function of ELL2 in the development of heart failure, we constructed ELL2 cardiomyocyte-specific knockout mice. Cardiomyocyte-specific Cre (NKX2.5-cre) mice were crossed with ELL2flox / flox mice to obtain NKX2.5-cre+; ELL2flox / flox (abbreviated as ELL2) CKO (This refers to) mice that can specifically knock out the ELL2 gene in cardiomyocytes. Figure 3 In wild-type (WT) mice, TAC surgery induces a heart failure phenotype characterized by significant decreased systolic function and ventricular dilation. Sham-operated ELL2 mice... CKO The cardiac function of mice was not significantly different from that of wild-type sham-operated mice, further demonstrating that ELL2 knockout does not affect cardiac function under physiological conditions. However, ELL2... CKOMice undergoing TAC surgery showed decreased systolic function and significantly inhibited ventricular dilation, indicating that ELL2 knockout under pathological conditions can protect cardiac function. Figure 3 BC.

[0131] Detecting the heart / fibula length ratio or heart / body weight ratio in mice indicates the ELL2 CKO The cardiac enlargement of mice after TAC surgery was significantly inhibited, see Figure 3 D(n=6).

[0132] Meanwhile, ELL2 CKO The survival of WT-TAC was significantly longer than that of WT-TAC (P = 0.0412), see [link to relevant documentation]. Figure 3 E in Chinese.

[0133] Results from qPCR detection of ANP and BNP, markers of cardiac hypertrophy, also indicate that ELL2 CKO The cardiac hypertrophy phenotype in -TAC mice was significantly suppressed, see Figure 3 In the middle F (n=6).

[0134] Example 4: Knocking out ELL2 in the heart of a heart in patients with heart failure can suppress the pathological phenotype of heart failure.

[0135] Four weeks after TAC surgery, wild-type mice and ELL2 mice were dissected. CKO In mouse hearts, ELL2 was found to be... CKO There was no significant difference in heart size between the -Sham sham surgery group and the WT-Sham sham surgery group. The heart size in the WT-TAC group was significantly larger than that in the WT-Sham group, while the ELL2 group... CKO Compared to the WT-TAC group, cardiac enlargement was inhibited in the -TAC group, see [reference needed]. Figure 4 HE staining results of cardiac sections also showed that the WT-TAC group had significantly greater left ventricular dilation compared to the WT-Sham group, while ELL2... CKO Left ventricular dilation was suppressed in the -TAC group compared to the WT-TAC group, see... Figure 4 B. This indicates that knocking out ELL2 in cardiomyocytes can inhibit left ventricular dilation.

[0136] Masson's tricom staining results also indicate that ELL2 CKO There was no significant difference in cardiac fibrosis between the -Sham group and the WT-Sham group. The WT-TAC group showed a significantly increased incidence of cardiac fibrosis compared to the WT-Sham group, while ELL2... CKO Compared with the WT-TAC group, cardiac fibrosis was significantly inhibited in the -TAC group, see... Figure 4 The CD count was 3. This indicates that knocking out ELL2 in cardiomyocytes can inhibit cardiac fibrosis.

[0137] WGA staining results also indicate that ELL2 CKO There was no significant difference in cardiomyocyte size between the WT-Sham and WT-Sham groups. The WT-TAC group showed a significantly larger cardiomyocyte cross-sectional area compared to the WT-Sham group, while the ELL2 group... CKO Compared with the WT-TAC group, cardiomyocyte hypertrophy was significantly inhibited in the -TAC group. Figure 4 The mean EF (n=4) indicates that knocking out ELL2 in cardiomyocytes can also inhibit cardiomyocyte hypertrophy.

[0138] TUNEL staining results also indicate that ELL2 CKO There was no significant difference in cardiac cell apoptosis between the WT-Sham group and the WT-Sham group. The WT-TAC group showed a significant increase in cardiac cell apoptosis compared to the WT-Sham group, while ELL2... CKO Compared with the WT-TAC group, the increase in cardiac cell apoptosis in the -TAC group was significantly inhibited. Figure 4 GH (n=6). This indicates that knocking out ELL2 in cardiomyocytes can also inhibit cardiac cell apoptosis.

[0139] Example 5: Cellular experiments demonstrate that knocking out ELL2 in cardiomyocytes can inhibit myocardial hypertrophy.

[0140] To further demonstrate the role of ELL2 in the development of myocardial hypertrophy or dilation, we extracted cardiomyocytes from the hearts of newborn mice for primary culture and induced cardiomyocyte hypertrophy by stimulation with angiotensin II (AngII, 1 μM) for 48 hours. Western blotting experiments showed that the expression of ELL2, an important component of the SEC complex, was significantly upregulated in cardiomyocytes after AngII stimulation. Figure 5 In addition, qPCR experiments showed that the expression of markers of cardiomyocyte hypertrophy was also significantly upregulated after AngII stimulation, see [reference needed]. Figure 5 B.

[0141] Extract ELL2 CKO Mouse cardiomyocyte culture experiments showed that, compared to wild-type mouse cardiomyocytes, ELL2 CKO In mice, cardiomyocytes showed a significant decrease in surface area after AngII stimulation. Figure 5 In the CD40-qPCR experiment, AngII stimulation of ELL2 cells was observed. CKO The expression of mastoid markers in mouse cardiomyocytes was also significantly upregulated, see [reference needed]. Figure 5 E. This indicates that knocking out ELL2 in cardiomyocytes can inhibit cardiomyocyte hypertrophy.

[0142] Example 6: RNA-Seq demonstrated that knocking out ELL2 in cardiomyocytes can inhibit signaling pathways such as cardiac hypertrophy, fibrosis, and inflammation in the development of heart failure.

[0143] To further elucidate the role of ELL2 in the development of heart failure, we extracted cardiac RNA from ELL2-overexpressing or knockout mice for transcriptome sequencing. Differential gene analysis showed that specific knockout of ELL2 in cardiomyocytes significantly altered the transcriptome of the heart in heart failure. (See below) Figure 6 Analysis of the ELL2 A.GO signaling pathway showed that conditional knockout of ELL2 significantly downregulated the expression of genes related to extracellular matrix and muscle structure development. Figure 6 Similarly, specific overexpression of ELL2 in cardiomyocytes can also cause significant changes in the transcriptome of the heart in heart failure, see [reference needed]. Figure 6 Analysis of the C.O signaling pathway showed that overexpression of ELL2 significantly upregulated the expression of genes related to extracellular matrix and muscle structure development. Figure 6 D.

[0144] GSEA analysis of differentially expressed genes showed that, compared to the WT-TAC group, ELL2 CKO The expression of genes related to cardiac hypertrophy, fibrosis, and inflammation was significantly downregulated in the hearts of mice in the WT-TAC group. In contrast, the expression of genes related to cardiac hypertrophy, fibrosis, and inflammation was significantly downregulated in the ELL2 group. OE The expression of genes related to cardiac hypertrophy, fibrosis, and inflammation was significantly upregulated in the hearts of mice in the TAC group. This indicates that conditional knockout of ELL2 in cardiomyocytes alleviates the pathological phenotype of heart failure by inhibiting the expression of genes related to cardiac hypertrophy, fibrosis, and inflammation. See Figure 7 .

[0145] qPCR results also showed that ELL2 knockout downregulated the expression of fibrosis-related genes in the heart, while ELL2 overexpression upregulated the expression of fibrosis-related genes. See Figure 8 .

[0146] Example 7: Knockout of ELL2 in cardiomyocytes inhibits SEC assembly

[0147] ELL2, a key component of the SEC complex, is upregulated in the heart of heart failure. Simultaneously, RNA polymerase II S2 phosphorylation is significantly increased, indicating increased transcriptional SEC assembly. Other SEC components, such as AFF4 and BRD4, showed no significant difference in expression between the sham-operated and TAC-related heart failure groups. Knockout of ELL2 in cardiomyocytes reduces ELL2 expression and RNA polymerase II S2 phosphorylation levels, while other SEC components, AFF4 and BRD4, are not significantly affected. Figure 9 This indicates that knocking out ELL2 in cardiomyocytes can inhibit the assembly of SEC in the heart of a heart failure patient, thereby affecting the transcription of heart failure-related genes and inhibiting the progression of heart failure.

[0148] Example 8: Drugs targeting the SEC complex can inhibit the assembly of the SEC complex in the heart.

[0149] Adult male C57BL / 6J mice (8 weeks old) were randomly divided into three groups. The experimental group underwent heart failure induced by transarterial chemoacupuncture (TAC). Two days after TAC, mice were randomly administered either the control solvent (TAC) or 20 mg / kg / day of KL-1 (TAC+KL-1). The control group was the sham-operated group. KL-1 was administered continuously for 4 weeks. Changes in cardiac function were assessed by echocardiography before TAC (D0), 2 weeks post-operation, and 4 weeks post-operation. Four weeks post-operation, the animals were sacrificed, and cardiac tissue was collected. Pathological features such as cardiac dilatation, cardiac fibrosis, and myocardial hypertrophy were detected using techniques including Western blotting, immunohistochemical staining, and qPCR. The experimental procedure is described below. Figure 10 A.

[0150] Immunoblot experiments showed that, compared to the sham-operated group, the expression of ELL2, the main component of the SEC complex, was upregulated in the TAC-operated group, but AFF4 remained unchanged. The key subunit CDK9 of pTEF-b also showed no significant change, and total RNA PolII remained unchanged, but phosphorylated RNA PolII S2 expression was upregulated. The small molecule KL-1, targeting AFF4 in the SEC complex, significantly reduced AFF4 levels and significantly inhibited the upregulation of ELL2 and RNA PolII S2 induced by TAC. These results indicate that KL-1 can inhibit SEC assembly, thereby inhibiting RNA PolII transcriptional activity. (See attached figures). Figure 10 B.

[0151] Example 9: Drugs targeting the SEC complex can inhibit the progression of heart failure.

[0152] Continuous echocardiography (ECHO) revealed no significant differences in cardiac function among the three groups of mice before surgery. Following TAC surgery, cardiac function in the mice significantly declined, as evidenced by a persistent decrease in ejection fraction (EF) and systolic function (FS) compared to the sham-operated group. Simultaneously, the left ventricular diameter (LVID) significantly increased, and the posterior wall of the left ventricle thickened significantly, exhibiting a phenotype of left ventricular hypertrophy combined with heart failure.

[0153] KL-1 treatment significantly inhibited the decline in cardiac function, as demonstrated by the fact that, compared to the untreated TAC group, the KL-1 treatment group significantly prevented the decrease in ejection fraction and the downregulation of systolic function, and also significantly prevented left ventricular dilation and left ventricular posterior wall thickening. These results indicate that KL-1 treatment can significantly inhibit the progression of left ventricular hypertrophy and heart failure. (See attached results). Figure 11 .

[0154] Example 10: Drugs targeting the SEC complex can inhibit the pathological progression of heart failure.

[0155] Four weeks after TAC surgery, mouse hearts were harvested and weighed. The results showed that compared to the sham-operated group, the TAC group mice had significantly enlarged hearts, while KL-1 treatment significantly inhibited the phenotype of cardiac hypertrophy. Specifically, compared to the TAC group, the KL-1 treatment group significantly inhibited the increase in heart weight and fibular length ratio (HW / TL). (See attached table). Figure 4 qPCR experiments also demonstrated that, compared to the sham-operated group, the expression of Nppa and Nppb genes, markers of cardiac hypertrophy, was significantly upregulated in the hearts of mice in the TAC group, while KL-1 treatment significantly inhibited the expression of Nppa and Nppb genes. (See results below.) Figure 12 AB in the middle.

[0156] Four weeks after TAC surgery, mouse heart tissue was collected for Masson's Trichrome staining to detect cardiac fibrosis. The results showed that compared to the sham-operated group, fibrosis was significantly increased in the hearts of mice in the TAC group, while KL-1 treatment significantly inhibited the development of fibrosis. (See attached figures). Figure 12 Medium CD.

[0157] Four weeks after TAC surgery, mouse heart tissue was collected for WGA (Invitrogen) staining to detect myocardial hypertrophy. The results showed that compared to the sham-operated group, the cross-sectional area of ​​cardiomyocytes in the TAC group was significantly increased, while KL-1 treatment significantly inhibited the increase in cardiomyocyte cross-sectional area. (See attached results). Figure 12 Medium EF.

[0158] qPCR experiments also demonstrated that, compared to the sham-operated group, the expression of cardiac hypertrophy markers and fibrosis genes in the hearts of TAC-treated mice was significantly upregulated, while KL-1 treatment significantly inhibited the expression of hypertrophy markers and fibrosis genes. (See results below.) Figure 13 .

[0159] Example 11: Drugs targeting the SEC complex can reverse the pathological progression of heart failure.

[0160] To further investigate the therapeutic effect of SEC-targeting small molecules in a mouse model of heart failure, we randomly assigned animals to receive either a control solvent or 20 mg / kg / day of KL-1 four weeks after transcatheter arterial compression (TAC). Cardiac function was assessed by echocardiography at 4, 6, and 8 weeks post-TAC. At 8 weeks post-TAC, hearts were harvested to examine pathological phenotypes such as cardiac hypertrophy. The experimental procedure is described below. Figure 14Echocardiography results showed that cardiac function was significantly reduced 4 weeks after TAC compared to the sham surgery group. At 4 weeks post-TAC, there was no difference in cardiac function between the TAC group and the TAC combined with KL-1 treatment group. However, at 6 weeks post-TAC, i.e., 2 weeks after KL-1 treatment, cardiac function was significantly improved in the KL-1 treatment group compared to the untreated group, manifested as increased ejection fraction and decreased left ventricular diameter. At 4 weeks after KL-1 treatment, cardiac function was also significantly improved in the KL-1 treatment group compared to the untreated group. This indicates that KL-1 treatment can still improve cardiac function in a heart failure model. (See attached table). Figure 14 B.

[0161] Eight weeks after TAC surgery, mouse hearts were harvested, and it was found that the hearts in the TAC group were significantly enlarged compared to the sham-operated group. KL-1 treatment significantly reversed the cardiac hypertrophy, as shown in the results below. Figure 15 This demonstrates that KL-1 treatment can still remodel the heart structure and improve cardiac hypertrophy in a model of existing heart failure.

[0162] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. The use of inhibitors of super-extended complex (SEC) in the preparation of drugs for the treatment of heart failure.

2. The application according to claim 1, wherein the inhibitor targets one or more of the SEC subunits.

3. The application according to claim 2, wherein the SEC subunit comprises: One or more of EAF1, EAF2, ELL1, ELL2, ELL3, AFF1, AFF2, AFF3, AFF4, AF9, ENL, and P-TEFb.

4. In the application according to claim 3, the inhibitor targets SEC subunits including P-TEFb, and targets CDK9 and / or cyclin T1 subunits therein.

5. The application according to claim 3, wherein the SEC subunit includes at least one or more of ELL2, AFF4, and P-TEFb.

6. The application according to any one of claims 2 to 5, wherein the inhibitor comprises at least one of antisense oligonucleotide, dsRNA, siRNA, shRNA, miRNA, RNAi, and ribozyme, which is designed to reduce or block the expression of the SEC subunit.

7. The application according to any one of claims 2 to 5, wherein the inhibitor comprises at least one of an antibody or an antigen-binding fragment thereof, a small molecule compound, or an aptamer, which is designed to reduce or counteract the activity of the SEC subunit and / or block the assembly of the SEC.

8. The application according to claim 1, wherein the inhibitor blocks the interaction between AFF4 and the P-TEFb subunit (particularly cyclin T1).

9. The application according to claim 8, wherein the inhibitor comprises KL-1 and / or KL-2.

10. The application according to any one of claims 2 to 5, 8, and 9, wherein the inhibitor knocks down or removes the gene of the SEC subunit or the gene regulating the expression of the SEC subunit by means of ZFN technology, TALEN technology, or CRISPR technology.

11. The application according to claim 10, wherein the inhibitor comprises gRNA and a CRISPR enzyme, or a vector capable of expressing them.

12. The application according to any one of claims 1 to 5, 8, 9, 11, wherein the inhibitor is used alone or in combination with other drugs.

13. The application according to claim 12, wherein the other drugs include: At least one of the following: diuretics, renin-angiotensin system inhibitors, vasodilators, aldosterone antagonists, β-adrenergic receptor blockers, MRA, sodium-glucose cotransporter 2 inhibitors, sinoatrial node pacemaker current inhibitors, rehmannia-based drugs, guanylate cyclase stimulants, and myosin agonists.

14. The application according to any one of claims 1 to 5, 8, 9, 11, 13, wherein the inhibitor and a pharmaceutically acceptable excipient constitute a pharmaceutical composition.

15. The application according to claim 14, wherein the form of the pharmaceutical composition is selected from one or more of the following: solution, injection, spray, nasal drops, aerosol, powder, tablet, capsule, and granule.

16. The application according to any one of claims 1 to 5, 8, 9, 11, 13, 15, wherein the heart failure is heart failure with preserved ejection fraction or heart failure with decreased ejection fraction.

17. The application according to any one of claims 1 to 5, 8, 9, 11, 13, and 15, wherein the efficacy of the drug includes at least one of inhibiting left ventricular dilation, inhibiting cardiac fibrosis, inhibiting cardiomyocyte hypertrophy, and inhibiting cardiac cell apoptosis.

18. The application according to any one of claims 1 to 5, 8, 9, 11, 13, 15, wherein the patient with the drug is a mammal.

19. The application according to claim 18, wherein the patient with the drug is a primate.

20. The application according to claim 19, wherein the patient with the drug is a human being.

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