WO2022027953A1 - C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用 - Google Patents

C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用 Download PDF

Info

Publication number
WO2022027953A1
WO2022027953A1 PCT/CN2021/076760 CN2021076760W WO2022027953A1 WO 2022027953 A1 WO2022027953 A1 WO 2022027953A1 CN 2021076760 W CN2021076760 W CN 2021076760W WO 2022027953 A1 WO2022027953 A1 WO 2022027953A1
Authority
WO
WIPO (PCT)
Prior art keywords
fibrosis
collagen
bumetanide
diseases
larp6
Prior art date
Application number
PCT/CN2021/076760
Other languages
English (en)
French (fr)
Inventor
余鹰
左胜锴
申毓军
崔辉
Original Assignee
天津医科大学
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 天津医科大学 filed Critical 天津医科大学
Publication of WO2022027953A1 publication Critical patent/WO2022027953A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
    • C07C311/39—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to the application of C188-9, Venetoclax and Bumetanide as medicines for preventing and treating fibrotic diseases.
  • Fibrosis plays an important role in diseases such as liver disease, kidney disease, idiopathic pulmonary fibrosis, and heart failure. Fibrosis is also a major pathological feature of many chronic autoimmune diseases, such as scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus.
  • Fibrosis also plays an important role in tumor invasion and metastasis, chronic rejection and many progressive myopathies.
  • the common feature of these diseases is the excessive accumulation of collagen, especially type I collagen, but there are few therapeutic targets specifically targeting the pathogenesis of fibrosis, and inhibition of collagen synthesis is a potential important target for the treatment of fibrosis-related diseases one of the points.
  • TGF- ⁇ transforming growth factor- ⁇
  • PDGF platelet growth factor
  • Fibroblasts play an important role in the fibrosis process. For example, after vascular intima injury, vascular smooth muscle cells and fibroblasts proliferate excessively, secrete a large amount of extracellular matrix including collagen, resulting in thickening of the vascular wall and stenosis of the vascular lumen, further leading to atherosclerosis, hypertension, The occurrence of various diseases such as myocardial infarction. Elucidating the molecular mechanisms of collagen synthesis in fibroblasts can not only deepen our understanding of fibrosis-related diseases, but also provide new therapeutic targets for anti-fibrosis.
  • TGF- ⁇ is the most important pro-fibrotic cytokine in various organs and a key molecule in the process of fibrosis. It has been reported that TGF- ⁇ signaling is associated with fibrosis in the liver, lung, kidney, skin, and heart, and that TGF- ⁇ 1 signaling can reduce the progression of many fibrosis models at the animal level. In recent years, the signaling pathway regulated by the synthesis and activity of TGF- ⁇ is a new hot target for the development of anti-fibrotic drugs. However, TGF- ⁇ signaling also plays an important role in other life activities. For example, mice deficient in TGF- ⁇ 1 develop various symptoms of autoimmune disorders and are more likely to develop tumors. Therefore, the TGF- ⁇ signaling pathway is not an ideal target for the treatment of fibrosis-related diseases.
  • Type I collagen is a heterotrimer composed of two ⁇ 1 and one ⁇ 2 peptide chains, and is the most abundant protein in the human body. Under normal circumstances, type I collagen is highly expressed in skin, bones and tendons, but low in parenchymal organs; once an organ is injured, the level of type I collagen increases rapidly in both transcriptional and post-transcriptional levels, and its protein expression level increases. It can reach 50-100 times the original expression, and participate in the repair process after organ damage.
  • mRNAs encoding type I collagen contain cis-acting elements in their 3' and 5' untranslated regions (UTRs) to control the stability of mRNAs.
  • UTRs 3' and 5' untranslated regions
  • 5'SL 5'stem loop
  • LARP6 (La ribonucleoprotein domain family member 6) is a member of the La-domain-containing ribonucleoprotein family, which has a conserved La domain and a highly variable RNA recognition sequence ( RNA recognition motif, RRM).
  • LARP6 is the only protein involved in the stability of type I collagen mRNAs among about 800 RNA-binding proteins discovered so far. It can bind to the 5'SL of type I and type III collagen mRNAs, thereby regulating the stability and translation of these mRNAs. Knockdown of LARP6 can greatly reduce the expression levels of type I collagen in mRNA and protein.
  • Insulin-like growth factor-1 can increase collagen synthesis by increasing the expression of LARP6, and overexpression of IGF-1 in Apoe-/- mice can increase the degree of fibrosis in atherosclerotic plaques, This signal also plays a role in regulating the synthesis of type I collagen during osteoblast differentiation.
  • the regulation of type I and type III collagen synthesis by LARP6 includes interaction with myosin filaments to regulate the secretion of type I collagen and interaction with Vimentin to increase the half-life of collagen mRNA, but LARP6 specifically regulates proteins and Whether the binding site of LARP6 to collagen can bind other molecules is still unclear and needs to be explored.
  • the purpose of the invention is to provide the application of C188-9, Venetoclax and Bumetanide as medicines for preventing and treating fibrotic diseases.
  • the functional product is a product or potential substance capable of treating, relieving, inhibiting and regulating the occurrence and development of fibrotic diseases in the body or a potential substance; the functional product is a single preparation or a combination comprising an effective amount of preparation ingredients thing.
  • the functional product targets the RRM region of LARP6 protein.
  • the functional product is used to play one or more of the following functions:
  • the fibrotic diseases include pulmonary fibrosis, liver fibrosis, renal fibrosis, cardiac fibrosis, endometrial fibrosis, eye fibrosis, pancreatic fibrosis, spleen fibrosis, bone marrow fibrosis or fibrosis chemical-induced diseases.
  • the pulmonary fibrosis disease is drug-induced pulmonary fibrosis
  • the drug-induced pulmonary fibrosis is sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis or radiation-induced pulmonary fibrosis, other pulmonary fibrosis of unknown etiology, and pulmonary fibrosis caused by pulmonary fibrosis liver fibrosis of viral hepatitis, alcoholic hepatitis, autoimmune disease, fatty liver, malnutrition, chronic congestive heart failure or drug-induced liver fibrosis, other liver fibrosis of unknown etiology , and diseases induced by liver fibrosis; hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, renal transplant rejection, pyelonephritis, kidney stones, hyperlipidemia, diabetes mellitus, hyperuricemia Renal fibrosis due to urine disease, hypercalciuria, other renal
  • the three compounds of C188-9, Venetoclax and Bumetanide involved in the present invention are more preferably selected from:
  • Venetoclax Cat. No.: T2119, CAS, 1257044-40-8, Targetmol (Wellesley Hills, MA, USA).
  • Bumetanide Cat. No.: T0108, CAS, 28395-03-1, Targetmol (Wellesley Hills, MA, USA).
  • the present invention mainly adopts advanced biological information and molecular biology technologies such as blood pressure telemetry technology, laser confocal microscopy technology, Real-time PCR technology, microscale thermophoresis (MST), RNA co-immunoprecipitation, and immunoblotting technology.
  • advanced biological information and molecular biology technologies such as blood pressure telemetry technology, laser confocal microscopy technology, Real-time PCR technology, microscale thermophoresis (MST), RNA co-immunoprecipitation, and immunoblotting technology.
  • RT-PCR was used to detect C188-9 (Cat. No.: T4650), Venetoclax (Cat. No.: T2119), AT7519 (Cat. No.: T1778) ) Bumetanide (Cat. No. T0108) and Anidulafungin (Cat. No. T6088), five small-molecule compounds, can reduce collagen mRNA levels at the cellular level.
  • the purified LARP6-RRM protein was used for direct interaction analysis with five small molecules, and it was found that C188-9, Venetoclax and Bumetanide could directly interact with LARP6-RRM.
  • Bumetanide was selected for further cell and animal fibrosis model validation. Whether in mouse fibroblast NIH 3T3 cells or human fibroblast MRC-5, Bumetanide can effectively inhibit the binding of LARP6 to collagen mRNA and reduce collagen expression. In a bleomycin-induced mouse model of pulmonary fibrosis, Bumetanide can effectively attenuate pulmonary fibrosis and collagen content in mice.
  • C188-9, Venetoclax and Bumetanide can effectively inhibit collagen synthesis.
  • the present invention can confirm that the three compounds C188-9, Venetoclax and Bumetanide can be used as potential drugs for preventing or treating fibrosis of body organs.
  • Figure 1 is a flow chart of compound screening.
  • This application starts from 5,370 small molecule compounds and screened out the top 50 compounds that are highly binding to LARP6-RRM (RNA recognition sequence of LARP6 protein) through virtual docking; There are 5 kinds of small molecule compounds that can better inhibit the main proteins of fibrosis (including collagen 1 and collagen 3, of which collagen 1 is abbreviated as Col 1, and its coding mRNAs are cola1 and col1a2; collagen 3 is abbreviated as Col 3, and its coding mRNA is co3a1); and then analyzed by microthermophoresis technique and found that C188-9, Venetoclax and Bumetanide have direct interaction with LARP6-RRM.
  • LARP6-RRM RNA recognition sequence of LARP6 protein
  • Figure 2 illustrates that five compounds have the effect of inhibiting collagen 1 and 3 synthesis.
  • the top fifty compounds with strong binding ability to LARP6-RRM were screened by virtual docking to treat NIH 3T3 fibroblasts. After 24 hours, RNA was extracted to detect the transcriptional expression levels of collagen 1 and collagen 3, the major fibrous proteins.
  • concentrations of the compounds were: T1100, 25 ⁇ M; T3S0175, 10 ⁇ M; TL0005, 10 ⁇ M; T2S1158, 10 ⁇ M; T6S1525, 10 ⁇ M; T0937, 5 ⁇ M; T1494, 10 ⁇ M; T2415, 1 ⁇ M; T3706, 1 ⁇ M; T6909, 10 ⁇ M; T6956, 1 ⁇ M; T3043, 5 ⁇ M; T1829, 2 ⁇ M; T4650, 10 ⁇ M; T2119, 1 ⁇ M; T2405, 20 ⁇ M; T0182L2, 10 ⁇ M; T2676, 2 ⁇ M; T0213, 15 ⁇ M; T3620, 10 ⁇ M; T4353, 10 ⁇ M; T3175, 10 ⁇ M;
  • Figure 2A shows the RNA expression level of the ⁇ 1 chain of collagen 1 (abbreviation: Col1a1)
  • Figure 2B shows the RNA expression level of the ⁇ 2 chain of collagen 1 (abbreviation: Col1a2)
  • Figure 2C shows the ⁇ 1 chain of collagen 3 (abbreviation: Col3a1) RNA expression level, *P ⁇ 0.05 vs control group.
  • FIG. 3 illustrates that C188-9, Venetoclax and Bumetanide three compounds have direct interaction with LARP6-RRM.
  • Figure 3B shows that Bumetanide has no interaction with MBP
  • Figure 3D shows that C188-9 has no interaction with MBP
  • Figure F shows that Venetoclax has no interaction with MBP.
  • Figure 4A, Figure 4B, and Figure 4C show the interaction mode of C188-9, Venetoclax and Bumetanide with LARP6-RRM, respectively.
  • LARP6-RRM protein is represented by a linearly connected gray sheet structure, and the three small molecule compounds and the amino acid residues interacting with them on the protein are represented by sticks. Small molecule compounds and interacting amino acid residues are shown in grey and dark grey, respectively; K184, L290, M293 and K294 in Figure 4A represent lysine 184, leucine 290, and 293 of LARR6-RRM protein, respectively Methionine and lysine at position 294; D242, I246, I291, M293 and P295 in Figure 4B represent aspartic acid at position 242, isoleucine at position 246, and isoleucine at position 291, respectively, in LARR6-RRM protein , methionine 293 and proline 295; M185, D242, I246, R249 and K294 in Figure 4C represent the 185 methionine, 242 aspartic acid, 246 isoleucine of LARR6-RRM protein, respectively acid,
  • Figure 5 illustrates that Bumetanide inhibits LARP6 binding to collagen 1 and collagen 3 mRNA.
  • NIH 3T3 fibroblasts were treated with 2 ⁇ M Bumetanide, harvested 24 hours later, and UV-crosslinked for 5 min, followed by RNA immunoprecipitation with LARP6 antibody. Afterwards, it was reverse transcribed into cDNA, and the mRNA levels of collagen 1 and 3 were detected by RT-PCR; the figure shows that Bumetanide can significantly reduce the binding of LARP6 to collagen mRNA. *P ⁇ 0.05 vs control group.
  • Figure 6 illustrates that Bumetanide attenuates bleomycin-induced pulmonary fibrosis in mice.
  • Figure 6A shows the collagen production with or without Bumetanide in the bleomycin-induced mouse pulmonary fibrosis model detected by Masson's trichrome staining, and the dark gray is collagen fibers (the arrow indicates the dark gray example) ;
  • Figure 6B shows the statistical results of collagen production;
  • Figure 6C measures hydroxyproline in the lung tissue of mice with pulmonary fibrosis;
  • hydroxyproline is one of the imino acids, a non-essential amino acid, and a collagen tissue It is one of the main components of collagen and is a unique amino acid in collagen, which can reflect the degree of tissue fibrosis.
  • Figure 7 illustrates that Bumetanide inhibits LARP6 binding to collagen 1 and collagen 3 mRNAs in human fibroblasts.
  • TGF- ⁇ 1 transforming growth factor- ⁇ 1, transforming growth factor- ⁇ 1
  • RNA immunoprecipitation RNA immunoprecipitation. Afterwards, it was reverse transcribed into cDNA, and the mRNA levels of collagen 1 and 3 were detected by RT-PCR.
  • Figure 7A, Figure 7B, Figure 7C can show that TGF- ⁇ 1 can increase the binding of LARP6 to the mRNAs of collagen 1 and collagen 3, while Bumetanide can significantly reduce the binding of LARP6 to the mRNAs of collagen 1 and collagen 3 and eliminate TGF- ⁇ 1. increase.
  • Figure 8 shows that Bumetanide inhibits collagen 1 and collagen 3 production in human fibroblasts.
  • FIGS. 8A-C are RT-PCR detection of mRNA levels of collagen 1 and 3 in human fibroblast MRC-5 treated with 2 ⁇ M Bumetanide and/or 10 ng/ml TGF- ⁇ 1.
  • Figure 8D shows the protein levels of collagen 1 and 3 in Bumetanide and/or TGF-beta1 treated human fibroblasts MRC-5. *P ⁇ 0.05 vs solvent group. #P ⁇ 0.01 vs control group.
  • Constant means that the involved amino acid sequence or nucleic acid sequence has a high similarity or identity with the original sequence, and can maintain the basic structure, biological activity or function of the original sequence, and can generally be replaced by similar amino acid residues or Allelic (degenerate codon) substitutions etc. are obtained.
  • a “variant” refers to an amino acid sequence or nucleic acid sequence having one or more amino acid or nucleotide changes, which may include insertions, deletions, or substitutions of amino acids or nucleotides in the amino acid sequence or nucleic acid sequence. Variants may have conservative changes, in which the replaced amino acid has similar structural or chemical properties to the original amino acid, such as a substitution between leucine and isoleucine, or non-conservative changes.
  • homologous includes complete homology and partial homology. When describing a polypeptide, protein or amino acid sequence, it means having the same or similar structure or function, or having a similar amino acid sequence; when describing a nucleic acid sequence, it means having similarity or complementary nucleic acid sequences, also including nucleic acid sequences embodied according to the codon preference of the organism of the construct; “homologous” has a relatively broad meaning in the present invention, for example, including sequences with a certain percentage of identity ( amino acid sequence or nucleic acid sequence), or include variants of the sequence.
  • “Derivative" when describing a polypeptide, protein or amino acid sequence refers to a related polypeptide, protein or amino acid sequence derived from the original polypeptide, protein or amino acid sequence, which has properties and activities similar to the original polypeptide, protein or amino acid sequence. or function, for example, the polypeptide, protein or amino acid sequence of the present invention includes derivatives such as (i) fusion of the mature polypeptide with another compound, or (ii) fusion or insertion of additional amino acid sequences (linker, linker, etc.) into the amino acid sequence.
  • Example 1 Screening of three compounds C188-9, Venetoclax and Bumetanide as a process for the treatment of fibrotic diseases
  • NIH 3T3 fibroblasts were treated with the small molecule compounds screened in 1), and the collagen mRNA changes were detected by Real-Time PCR;
  • the Dosin Bioclassic known activity library (Cat. No.: L4000) is used to conduct computer virtual docking screening of 5370 compounds with biological activity that can cause biological responses in cells, tissues and even individuals.
  • the qPCR method can use up to 1 ⁇ g of Total RNA
  • Reaction procedure 95°C for 5 min to complete the pre-denaturation reaction; 40 rounds of PCR amplification, each round of reaction was incubated at 95°C for 25s to denature the template, incubated at 60°C for 25s to promote the pairing of primers and templates, and completed at 72°C for 30s Extend the reaction and record fluorescence readings. The results are shown in Figures 2A-2C.
  • MBP-LARP6 protein and MBP control protein were fluorescently labeled with NT 647 labeling kit (NanoTemper Technologies, Munchen, Germany).
  • the labeled MBP-LARP6 protein was incubated at a constant concentration (1 ⁇ M), and the concentration of ligands (different small molecule compounds) was serially diluted twice to 16 different concentrations (from 1 ⁇ M to 0.03nM).
  • Fluorescence analysis was performed on a Monolith NT.115 device (NanoTemper Technology, Munchen, Germany). MST power is 20% and excitation power is 75%. The measurement scheme is: 5s pre-fluorescence, 20s MST, 5s post-fluorescence, and 25s delay. Kd values were calculated by NT analysis software (NanoTemper Technologies).
  • Example 2 Bumetanide inhibits bleomycin-induced pulmonary fibrosis in mice.
  • the RT-PCR method is the same as the PCR detection in Example 1, and the results are shown in Figure 5.
  • mice to be used were anesthetized by intraperitoneal injection of Avertin.
  • mice mandibular glands were peeled sideways with cotton swabs to expose the trachea.
  • Bleomycin was injected into the mouse trachea at a dose of 3.5 mg/kg (dissolved in physiological saline) using an insulin syringe.
  • Bumetanide was subcutaneously injected at a dose of 2 mg/kg, and the control group was injected with the same amount of DMSO. 14 consecutive days of injections.
  • mice After 14 days, the mice were sacrificed, and the lung tissue was removed for paraffin embedding or hydroxyproline assay.
  • Example 3 Bumetanide inhibits collagen synthesis in human fibroblasts.
  • the present invention starts from the natural active compound library, through computer simulation screening, and repeated studies at the cellular animal level, it is found that in C188-9, Venetoclax and Bumetanide can be referred to as a new potential for clinical treatment of fibrotic diseases. drug.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Diabetes (AREA)
  • Cardiology (AREA)
  • Endocrinology (AREA)
  • Pulmonology (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Reproductive Health (AREA)
  • Hematology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Ophthalmology & Optometry (AREA)
  • Rheumatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用，从临床出发，以LARP6蛋白的RNA识别序列(RNA recognition motif,RRM)区域为对接目标，经在计算机虚拟对接模拟筛选，最后经过细胞以及动物实验，发现在损伤诱导的纤维化的发生发展过程中，C188-9，Venetoclax和Bumetanide(临床上常用的一种强力速效利尿剂)，通过结合LARP6蛋白的RRM区域，从而抑制胶原mRNA的稳定性来抑制胶原产生，进而抑制纤维化相关的病症。丰富了对纤维化相关疾病发病机理的基础研究的内容，为更深入探索纤维化疾病发病的本质原因提供了新的思路。同时，C188-9，Venetoclax和Bumetanide可参考作为临床上治疗纤维化疾病的一个新的潜在药物。

Description

C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用 技术领域
本发明创造属于生物技术领域，具体涉及C188-9、Venetoclax、Bumetanide作为预防和治疗纤维化疾病药物中的应用。
背景技术
器官纤维化的主要病理改变为器官组织内纤维结缔组织增多，实质细胞减少，持续纤维化可导致器官正常的结构破坏和生理功能减退，乃至衰竭，严重威胁人类健康和生命。纤维化在肝脏疾病、肾脏疾病、特发性肺纤维化和心脏衰竭等疾病中扮演重要的角色。纤维化也是许多慢性自身免疫性疾病的主要病理特征，比如硬皮病、风湿性关节炎、克罗恩病、溃疡性结肠炎、骨髓纤维化和系统性红斑狼疮等。纤维化还在肿瘤的侵袭转移、慢性排斥反应和许多渐进性肌病的过程中发挥重要作用。这些疾病的共同特征是胶原蛋白，特别是I型胶原的过度累积，但目前专门针对纤维化发病机制的治疗靶点还很少，抑制胶原蛋白的合成是潜在的治疗纤维化相关疾病的重要靶点之一。
1.胶原与组织纤维化
当机体器官受到损伤或应激后，适度的纤维化有助于损伤的修复，而过度纤维化则会加重疾病的发展。TGF-β(转化生长因子-β)和PDGF(血小板生长因子)信号主要是通过介导成纤维细胞过度分泌细胞外基质，在纤维化的过程中扮演着重要的角色。在这些细胞外基质中，I型胶原占比例达到80％-90％，在纤维化进程中起主导作用；Ⅲ型胶原也是纤维化中细胞外基质的成分，但所占比例较小；其他类型胶原如Ⅱ型，Ⅵ型等胶原则在纤维化基本检测不到。因此，抑制Ⅰ型胶原的合成可以有效地减少纤维化。成纤维细胞在纤维化过程中发挥着重要的功能。比如在血管内膜损伤后，血管平滑肌细胞以及成纤维细胞过度增殖，分泌包括胶原在内的大量细胞外基质，造成血管壁变厚以及血管腔狭窄，进一步导致如动脉粥样硬化，高血压，心肌梗死等多种疾病的发生。阐明成纤维细胞中胶原合成的分子机制不仅可以加深我们对纤维化相关疾病的理解，还可以为抗纤维化提供新的治疗靶点。
2.LARP6在胶原合成中的作用
目前认为，TGF-β是多种器官中最主要的促纤维化的细胞因子和纤维化进程中的关键分子。有报道指出，TGF-β信号与肝、肺、肾脏、皮肤和心脏的纤维化有关，并且在动物水平上TGF-β1信号通路可以减少许多纤维化模型的进展。近年来，TGF-β的合成与活性调节的信号通路是抗纤维化药物研发的新的热门靶点。但是，TGF-β信号在其他生命活动中也发挥重要功能，比如TGF-β1缺失的小鼠会出现多种自身免疫失调症状以及更容易发生肿瘤。因此，TGF-β信号通路并不是治疗纤维化相关疾病的理想靶点。
关于胶原转录后的调控的研究甚少，目前只有报道指出LARP6可以作为RNA结合蛋白，来增加胶原mRNA的稳定性，进而影响胶原蛋白的合成。Ⅰ型胶原蛋白是由两个α1和一个α2肽链组成异三聚体，是人体内含量最高的蛋白。正常情况下，Ⅰ型胶原在皮肤，骨骼和肌腱中高表达，而在实质性的器官中低表达；器官一旦受到损伤，Ⅰ型胶原在转录和转录后的水平都迅速升高，其蛋白表达量可达到原来表达量的50-100倍，参与到器官损伤后的修复过程中。病理情况下，Ⅰ型胶原合成的增多除了在转录水平外，更多的是转录后的mRNA的稳定性方面的调节。编码Ⅰ型胶原的mRNAs在其3’和5’非编码区域(untranslated regions,UTRs)含有顺式作用元件来控制着mRNAs的稳定性。在所有脊椎动物的Ⅰ型胶原的mRNAs的5’UTR区域，都有一个独立的5’颈环结构(5’stem loop,5’SL)，这一结构还存在于编码Ⅲ型胶原的mRNA中。LARP6(La ribonucleoprotein domain family member 6)是含有La结构域(La-domain)的核糖核蛋白家族中的一个成员，该蛋白家族都有一个保守的La结构域和一个高度可变的RNA识别序列(RNA recognition motif,RRM)。LARP6是目前发现的约800个RNA结合蛋白中唯一一个参与到Ⅰ型胶原mRNAs稳定性的蛋白，它可以与Ⅰ型和Ⅲ型胶原mRNAs 5’SL结合，进而调节这些mRNAs的稳定性和翻译。敲降LARP6可以很大程度上减少Ⅰ型胶原在mRNA和蛋白方面的表达水平。胰岛素样生长因子-1(IGF-1)可以通过增加LARP6的表达来增加胶原的合成，在Apoe-/-的小鼠中过表达IGF-1可以增加动脉粥样斑块中的纤维化程度，这一信号还在调节成骨细胞的分化过程中的Ⅰ型胶原的合成中发挥作用。LARP6对Ⅰ型和Ⅲ型胶原合成的调节，包括与肌球蛋白丝相互作用来调节Ⅰ型胶原的分泌以及与波形蛋白(Vementin)的相互作用来增加胶原mRNA的半衰期，但LARP6具体调节蛋白以及LARP6与胶原的结合位点是否可以结合其他分子仍不清楚，需要探究。
发明内容
本发明创造的目的在于提供C188-9、Venetoclax、Bumetanide作为预防和治疗纤维化疾病药物中的应用。
本发明的C188-9、Venetoclax、Bumetanide中的至少一化合物在制备治疗或预防机体器官纤维化疾病的功能产品方面的应用；
其中，C188-9(式I)、Venetoclax(式Ⅱ)、Bumetanide(式Ⅲ)的结构式分别为：
Figure PCTCN2021076760-appb-000001
其中，所述功能产品为能够对机体器官纤维化疾病的发生、发展产生治疗、缓解、抑制、调节的有益作用的产品或潜在物质；所述功能产品为单一制剂或包含有效量制剂成分的组合物。
其中，所述功能产品以LARP6蛋白的RRM区域为靶标。
其中，所述功能产品用于起到以下一种或多种作用：
i)降低成纤维细胞中的胶原的表达；
ii)抑制成纤维细胞中的LARP6与胶原mRNA的结合。
其中，所述纤维化疾病包括肺纤维化、肝纤维化、肾纤维化、心纤维化、子宫内膜纤维化、眼纤维化、胰腺纤维化、脾纤维增生疾病、骨髓纤维化疾病或由纤维化诱发的疾病。
进一步，所述肺纤维化疾病是药物性肺纤维化，药物性肺纤维化是结节病、尘肺、过敏性肺炎或放射线诱发的肺纤维化，其他病因未明的肺纤维化，以及由肺纤维化诱发的疾病；所述肝纤维化是病毒性肝炎、酒精性肝炎、自身免疫性疾病、脂肪肝、营养不良、慢性充血性心力衰竭或药物引起的肝脏纤维化，其他病因未明的肝脏纤维化，以及由肝纤维化诱发的疾病；所述肾纤维化是高血压、肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥、肾盂肾炎、肾结石、高血脂、糖尿病、高尿酸尿症、高钙尿症引起的肾脏纤维化，其他病因未明的肾脏纤维化，以及由肾纤维化诱发的疾病；所述心纤维化是缺血性心脏疾病、高血压、病毒性心肌炎、代谢性心肌病、克山病、扩张性心肌病、肥厚性心肌病、限制性心肌病或心律 失常引起的心脏纤维化、心脏重构和心肌肥厚，其他病因未明的心纤维化，以及由心纤维化诱发的疾病；所述子宫内膜纤维化是不同原因引起的子宫内膜纤维化病变，以及由子宫内膜纤维化诱发的疾病；所述眼纤维化是眼睛外伤、眼睛手术或糖尿病引起的眼视网膜纤维增生疾病，以及由眼纤维化诱发的疾病。
另外，本发明中涉及的C188-9，Venetoclax和Bumetanide三种化合物较优选自：
C188-9：货号：T4650，CAS，432001-19-9，Targetmol(Wellesley Hills,MA,USA)。
Venetoclax：货号：T2119，CAS，1257044-40-8，Targetmol(Wellesley Hills,MA,USA)。
Bumetanide：货号：T0108，CAS，28395-03-1，Targetmol(Wellesley Hills,MA,USA)。
本发明主要采用血压遥测技术，激光共聚焦显微技术、Real-time PCR技术、微量热泳动(microscale thermophoresis,MST)、RNA免疫共沉淀、免疫印迹技术等先进的生物信息和分子生物学技术手段，从胶原调节关键蛋白LARP6着手对LARP6在胶原稳定性中的作用进行了研究。首先以陶素生物已知活性的小分子库为筛选对象，以LARP6蛋白的RRM区域为对接目标，通过高通量虚拟对接筛选，筛选出能与LARP6的RRM区域结合力强的分子。再次，选出对接分数高的前五十小分子化合物，处理NIH 3T3成纤维细胞系，通过RT-PCR检测发现C188-9(货号：T4650)，Venetoclax(货号：T2119)，AT7519(货号：T1778)Bumetanide(货号：T0108)和Anidulafungin(货号：T6088)五种小分子化合物可在细胞水平降低胶原的mRNA水平。而后，进一步通过MST技术，用纯化的LARP6-RRM蛋白与五种小分子进行直接相互作用分析，发现C188-9，Venetoclax和Bumetanide三种化合物可以与LARP6-RRM直接相互作用。最后我们选取Bumetanide进行进一步的细胞和动物纤维化模型验证。无论是在小鼠的成纤维细胞NIH 3T3细胞还是人的成纤维细胞MRC-5中，Bumetanide可以有效抑制LARP6与胶原mRNA的结合，降低胶原蛋白的表达。在博来霉素诱导的小鼠肺纤维化模型中，Bumetanide可以有效减轻小鼠的肺纤维化和胶原含量。
本发明通过与LARP6对接发现C188-9、Venetoclax和Bumetanide三种化合物均可以有效抑制胶原蛋白合成。通过以上研究，本发明可以确认C188-9，Venetoclax和Bumetanide三种化合物可以作为预防或治疗机体器官纤维化的潜在药物。
附图说明
图1为化合物筛选流程图。
本申请从5370种小分子化合物出发，经过虚拟对接筛选出与LARP6-RRM(LARP6蛋白的RNA识别序列)相互高度结合的前五十种化合物；然后用这50种化合物，处理成纤维细胞，发现有5种小分子化合物有较好的抑制纤维化主要蛋白(包括胶原1和胶原3，其中胶原1简称为Col 1，其编码mRNA为cola1和col1a2；胶原3简称为Col 3，其编码mRNA为co3a1)的作用；再通过微量热泳动技术分析发现C188-9，Venetoclax和Bumetanide与 LARP6-RRM有直接相互作用。
图2说明五种化合物具有抑制胶原1和3合成的作用。
用虚拟对接筛选出与LARP6-RRM结合力强的前五十种化合物处理NIH 3T3成纤维细胞，24小时后，抽取RNA，检测纤维主要蛋白—胶原1和胶原3的转录表达水平。化合物的浓度分别是：T1100,25μM；T3S0175,10μM；TL0005,10μM；T2S1158,10μM；T6S1525,10μM；T6S1784,25μM；T1441,10μM；T4S0590,10μM；T4S2164,10μM；T6S1141,20μM；T4A2458,5μM；T0937,5μM；T1494,10μM；T2415,1μM；T3706,1μM；T6909,10μM；T6956,1μM；T1823,10μM；T2660,1μM；T1091,10μM；T4619,10μM；T6591,10μM；T3464,30μM；T5032,10μM；T3043,5μM；T1829,2μM；T4650,10μM；T2119,1μM；T2405,20μM；T0182L2,10μM；T3181,10μM；T0239,50μM；T1778,2μM；T3078,10μM；T0108,2μM；T2126,2μM；T2676,2μM；T0213,15μM；T3620,10μM；T4353,10μM；T3175,10μM；T2377,10μM；T6205,1μM；T0137,10μM；T1325,10μM；T0749,10μM；T3156,25μM；T6152,10μM；T3193,10μM；T6088,10μM；T7079,10μM。其中图2A是胶原1的α1链(简称：Col1a1)的RNA表达水平，图2B是胶原1的α2链(简称：Col1a2)的RNA表达水平，图2C是胶原3的α1链(简称：Col3a1)的RNA表达水平，*P<0.05vs control组。
图3说明C188-9，Venetoclax和Bumetanide三种化合物与LARP6-RRM有直接相互作用。
表达纯化带MBP(Maltose-Binding Protein，麦芽糖结合蛋白，是一种常用的蛋白标签)标签的LARP6-RRM蛋白以及对照MBP蛋白，将荧光标记的图中筛选到的五种小分子分别与MBP-LARP6或者MBP蛋白通过微量热泳动仪器检测相互作用。图3A说明Bumetanide与MBP-LARP6有相互作用，其平衡解离常数Kd＝0.848μM，图3B说明Bumetanide与MBP没有相互作用；图3C说明C188-9与MBP-LARP6有相互作用，其平衡解离常数Kd＝12.5μM，图3D说明C188-9与MBP没有相互作用；图E说明Venetoclax与MBP-LARP6有相互作用，其平衡解离常数Kd＝4.53μM，图F说明Venetoclax与MBP没有相互作用。
图4A、图4B、图4C分别体现了C188-9，Venetoclax和Bumetanide三种化合物与LARP6-RRM相互作用的模式。
其中，LARP6-RRM蛋白质由线状连接的灰色片状结构表示，三种小分子化合物以及蛋白上与之相互作用的氨基酸残基以棒状表示。小分子化合物和相互作用的氨基酸残基分别为灰色和深灰色；图4A中K184,L290,M293和K294分别代表LARR6-RRM蛋白第184位赖氨酸，第290位亮氨酸，第293位蛋氨酸和第294位赖氨酸；图4B中D242,I246,I291,M293和P295分别代表LARR6-RRM蛋白第242位天冬氨酸，第246位异亮氨酸，第291位异亮氨酸，第293位蛋氨酸和第295位脯氨酸；图4C中M185,D242,I246,R249和K294分别代表LARR6-RRM蛋白第185位蛋氨酸，第242位天冬氨酸，第246位异亮氨酸，第249位精氨酸和第294位赖氨酸。
图5说明Bumetanide抑制LARP6结合胶原1和胶原3的mRNA。
用2μM Bumetanide处理NIH 3T3成纤维细胞，24小时后收取细胞，紫外交联5min后，用LARP6抗体进行RNA免疫沉淀。之后，反转录为cDNA，RT-PCR检测胶原1和3的mRNA水平；图中显示Bumetanide可显著减少LARP6与胶原mRNA的结合。*P<0.05vs对照组。
图6说明Bumetanide可减轻博来霉素诱导的小鼠肺纤维化。
其中，图6A为通过马松三色染色检测博来霉素诱导的小鼠肺纤维化模型中，用或者不用Bumetanide后的胶原产生情况，深灰色的为胶原纤维(箭头指示为深灰色示例)；图6B对胶原产生情况进行的统计结果；图6C对肺纤维化小鼠的肺组织进行羟脯氨酸测定；羟脯氨酸是亚氨基酸之一，是一种非必需氨基酸，是胶原组织的主要成分之一，且为胶原中特有的氨基酸，可反应组织的纤维化程度。
图7说明Bumetanide可以在人的成纤维细胞中抑制LARP6结合胶原1和胶原3的mRNA。
用2μM Bumetanide和/或10ng/ml TGF-β1(transforming growth factor-β1,转化生长因子-β1)处理人成纤维细胞MRC-5，24小时后收取细胞，紫外交联5min后，用LARP6抗体进行RNA免疫沉淀。之后，反转录为cDNA，RT-PCR检测胶原1和3的mRNA水平。图7A、图7B、图7C可显示TGF-β1可增加LARP6与胶原1及胶原3的mRNA的结合， 而Bumetanide可显著减少LARP6与胶原1及胶原3的mRNA的结合并消除TGF-β1带来的增加。*P<0.05vs溶剂组。#P<0.01vs对照组。
图8体现Bumetanide在人的成纤维细胞中抑制胶原1和胶原3的产生。
其中，图8A-C为RT-PCR检测2μM Bumetanide和/或10ng/ml TGF-β1处理人成纤维细胞MRC-5中的胶原1和3的mRNA水平。图8D显示Bumetanide和/或TGF-β1处理人成纤维细胞MRC-5中的胶原1和3的蛋白水平。*P<0.05vs溶剂组。#P<0.01vs对照组。
具体实施方式
下面通过结合具体实施例对本发明创造进行进一步说明。
本说明书和权利要求书中使用的下列术语，除非另外说明具有下述一般含义，且下述含义被认为在本领域技术人员的知识范围之内：
“保守”指所涉及的氨基酸序列或核酸序列与原始序列具有较高的相似性或同一性，能够维持其原始序列基本的结构、生物学活性或功能，一般可以通过相似的氨基酸残基替换或等位基因(简并密码子)替换等获得。
“变体”指具有一个或多个氨基酸或核苷酸改变的氨基酸序列或核酸序列，所述改变可包括氨基酸序列或核酸序列中氨基酸或核苷酸的插入、缺失或替换。变体可具有保守性改变，其中替换的氨基酸与原氨基酸具有相似的结构或化学性质，如亮氨酸和异亮氨酸之间的替换，也可具有非保守性改变。
“同源”包括完全同源和部分同源，在描述多肽、蛋白质或氨基酸序列时，指具有相同或相似的结构或功能，或具有相似的氨基酸序列；在描述核酸序列时，指具有相似性或互补的核酸序列，也包括根据构建物的生物体的密码子偏爱性体现的核酸序列；“同源”在本发明具有相对较为广泛的含义，例如，包括具有一定百分比的相同性的序列(氨基酸序列或核酸序列)，或包括序列的变体。
“衍生物”在描述多肽、蛋白质或氨基酸序列时，指由原多肽、蛋白质或氨基酸序列衍生而来的关联多肽、蛋白质或氨基酸序列，其具有与原始多肽、蛋白质或氨基酸序列相似的性质、活性或功能，例如，本发明中多肽、蛋白质或氨基酸序列包括这样的衍生物：(i)成熟多肽与另一种化合物融合，或者(ii)在氨基酸序列中融合或插入附加的氨基酸序列(linker、蛋白质纯化标识序列、酶切位点等)；等；在描述核酸序列时，指由原始序列衍生未来的关联序列，其具有与原始核酸序列相似的性质、活性或功能，可以包括：(i)序列或基因中连续或间隔地插入、缺失、替换一个或多个碱基(优选为等位基因的替换)，且所述一个或 多个氨基酸残基的插入、缺失、替换在同一序列或基因中可同时或不同时存在；(ii)序列或基因中一个或多个碱基被修饰；(iii)序列或基因中融合或插入编码附加的氨基酸序列的基因；等。
下面通过具体实验及分析和讨论详细阐述C188-9，Venetoclax和Bumetanide三种化合物与纤维化疾病之间的相关性。
实施例1 筛选C188-9，Venetoclax和Bumetanide三种化合物作为治疗纤维疾病的过程
本实施例涉及的主要实施过程如下：
1)计算机模拟筛选出与LARP6-RRM亲和力强的五十种小分子化合物；
2)用1)中筛选到的小分子化合物处理NIH 3T3成纤维细胞，Real-Time PCR检测胶原mRNA变化；
3)用2)中筛选到的影响胶原合成的小分子化合物，用微量热泳动检测小分子化合物与胶原的直接结合；
本实施例涉及的主要实验部分如下：
1、筛选出与LARP6-RRM亲和力强的小分子化合物
实验步骤如下：
利用陶素生物经典已知活性库(货号：L4000)是5370个具有生物活性，能引起细胞、组织甚至个体生物学反应的化合物进行计算机虚拟对接筛选。
2、Real-Time PCR检测胶原表达
实验步骤如下：
1)将上述处理的NIH 3T3成纤维细胞加Trizol后，室温放置5min，使其充分裂解(注：此时可放入-70℃长期保持)；
2)按200μl氯仿/ml Trizol加入氯仿，振荡混匀15s，室温放置10min(注：禁用漩涡振荡器，以免基因组DNA断裂)；
3)将样品于4℃条件下12000g离心15min；
4)小心吸取上层水相转移至新的1.5ml离心管(RNase free)中；
5)按0.5ml异丙醇/ml Trizol加入异丙醇，充分混匀，于室温放置5-10min；
6)将样品于4℃12000g离心10min，弃上清，RNA沉于管底；
7)按1ml 75％乙醇/ml Trizol加入75％乙醇(DEPC处理后的去离子水配制)，温和振荡离心管，悬浮洗涤沉淀；
8)将样品于4℃8000g离心5min，尽量弃上清；
9)室温晾干或真空干燥5-10min。注：RNA样品不要过于干燥，否则很难溶解；
10)可用20-50μl DEPC H 2O溶解RNA样品，55-60℃5-10min；
11)Nano Drop测量RNA浓度；
12)逆转录及RT-PCR反应条件：参照Takara公司反转录试剂盒说明书完成。
基因组DNA的除去反应体系如下表所示：
表1 基因组DNA的除去反应体系
Figure PCTCN2021076760-appb-000002
置于42℃反应2min(或室温5min *b)。
*a：20μl反转录反应体系中，
Figure PCTCN2021076760-appb-000003
qPCR法最多可使用1μg的Total RNA；
*b：室温反应时，可以延长至30分钟；
反转录反应体系如下表所示：
表2 反转录反应体系
Figure PCTCN2021076760-appb-000004
置于37℃反应15min，继以85℃反应5s。
定量PCR反应体系如下表所示：
表3 定量PCR反应体系
Figure PCTCN2021076760-appb-000005
反应程序：95℃反应5min完成预变性反应；PCR循环扩增40轮，每轮反应于95℃保温25s以使模板变性、于60℃保温25s促进引物与模板配对结合、于72℃反应30s完成延伸反应并记录荧光读值。结果见图2A-2C。
3、微量热泳动检测蛋白与小分子的相互作用
实验步骤如下：
1)利用AKTA快速蛋白液相色谱系统纯化重组蛋白MBP-LARP6蛋白或MBP对照蛋白，使蛋白溶解在20mM HEPES(pH 7.5)和100mM NaCl缓冲液中。
2)使用NT 647标记试剂盒(NanoTemper Technologies,Munchen,Germany)对MBP-LARP6蛋白和MBP对照蛋白进行荧光标记。
3)测定在含有20mM HEPES(pH 7.5)、100mM NaCl和0.05％Tween-20的缓冲液中进行。
4)经过短暂的孵化期后，样品被装入MST NT.115标准玻璃毛细管中。
5)每次实验前，均确认试样未粘附在毛细管壁上，试样未发生聚集现象。
6)标记的MBP-LARP6蛋白孵育在恒定浓度(1×M)下，配体(不同小分子化合物)浓度连续稀释两倍至16个不同浓度(从1×M到0.03nM)。
7)十六种滴定系列配体与标记的MBP-LARP6混合生成最终的反应混合物。
荧光分析在Monolith NT.115装置(NanoTemper技术，Munchen，德国)。MST功率为20％，励磁功率为75％。测量方案为:5s前荧光，20s MST,5s后荧光，延迟25s。Kd值由NT分析软件(NanoTemper Technologies)计算。
结果见图3A-F.
小结：在本实施例的上述实验过程中，我们通过对陶素生物已知活性的小分子库进行筛 选，以LARP6蛋白的RRM区域为对接目标，通过高通量虚拟对接筛选，筛选出能与LARP6的RRM区域结合力强的分子。再次，选出对接分数高的前五十小分子化合物，处理NIH 3T3成纤维细胞系，通过RT-PCR检测发现C188-9(货号：T4650)，Venetoclax(货号：T2119)，AT7519(货号：T1778)Bumetanide(货号：T0108)和Anidulafungin(货号：T6088)五种小分子化合物在细胞水平降低胶原1及胶原3的mRNA水平。而后，进一步通过MST技术，用纯化的LARP6-RRM蛋白与五种小分子进行直接相互作用分析，发现C188-9，Venetoclax和Bumetanide三种化合物可以与LARP6-RRM直接相互作用。因此，我们推测C188-9，Venetoclax和Bumetanide三种化合物化合物具有抗纤维化的作用，后续试验我们选取Bumetanide作为代表分子，在小鼠的肺纤维化模型以及人的成纤维细胞中进行验证。
实施例2 Bumetanide抑制博来霉素诱导的小鼠肺纤维化。
本实施例涉及的主要实验部分如下：
1、RIP-PCR检测胶原基因表达
1)用Bumetanide或者溶剂对照处理好细胞。
2)将10cm培养皿中的细胞置于冰中交联，在1ml冷PBS的作用下，400mJ/cm2的UV照射5分钟。
3)弃PBS，加入含200U/ml RNase抑制剂(Takara)和蛋白酶抑制剂鸡尾酒(Roche)的500ul冷洗缓冲液(1×PBS,0.1％SDS,0.5％NP-40,0.5％脱氧胆酸钠)，冰敷10分钟。
4)刮去细胞，放入1.5ml管中。
5)4℃16000g离心20分钟。
6)加入RQ I(Promega)至终浓度为1U/mol/l,37℃水浴孵育5分钟，然后在冰上冷却5分钟。
6)对于免疫共沉淀，用5ul抗体或对照IgG在摩天轮上4℃孵育过夜。
7)免疫沉淀与蛋白A或蛋白G珠在4℃下孵育3小时。
8)在RIP缓冲液(20mM Tris-Cl,pH 7.4,2000mM NaCl,1mM EDTA,0.3Triton X-100,5％甘油)中，4℃洗涤4次。
9)使用TRIzol(Invitrogen)回收RNA。
RT-PCR方法同实施例1中的PCR检测，结果见图5。
2、小鼠肺纤维化模型建立
1)取6-8周成年雄鼠，随机分为两组。
2)待用小鼠通过腹腔注射阿弗汀进行麻醉。
3)对小鼠颈部用脱毛膏进行脱毛处理，随后用75％医用酒精对脱毛部位进行消毒处理。
4)用消毒的手术刀沿颈部中线划开2～3cm的小口，暴露出下颌腺。
5)用棉签将小鼠下颌腺向两侧剥开，暴露出气管。
6)用胰岛素注射器按照3.5mg/kg(溶于生理盐水中)的剂量将博来霉素注射入小鼠气管中。
7)用5-0真丝医用缝合线缝合伤口。
8)按照2mg/kg的剂量皮下注射Bumetanide，对照组注射等量的DMSO。连续注射14天。
9)14天后，牺牲小鼠，取出肺组织进行石蜡包埋或者进行羟脯氨酸测定。
3、组织切片马松三色染色
1)将中性甲醛溶液固定肺组织，石蜡切片4～6μm。
2)肺组织石蜡切片依次置于二甲苯Ⅰ15min→二甲苯Ⅱ15min→二甲苯：无水乙醇＝1:1 2min；然后置于100％乙醇Ⅰ5min→100％乙醇Ⅱ5min→80％乙醇5min→蒸馏水5min。
3)Bouin液固定10～15min。
4)Harris苏木素染色4～5min，流水冲洗2min。
5)0.5％盐酸酒精分化10～30s，流水冲洗5min。
6)Masson复合染色液4～5min。
7)0.2％醋酸水溶液稍冲洗。
8)5％磷钼酸分化5～10min。
9)0.2％醋酸水溶液稍冲洗。
10)2％苯胺蓝溶液10～30s。
11)无水乙醇冲洗干净，自然晾干，中性树胶封片。
12)显微镜选取视野拍摄分析组织重塑,结果见图6A。
13)使用imageJ软件进行统计胶原面积，结果见图6B。
小结：以上结果说明Bumetanide可以减少LARP6与胶原mRNA的结合(图5)，以及有效缓解博来霉素诱导的小鼠肺纤维化模型(图6)。
实施例3 Bumetanide抑制人成纤维细胞中的胶原合成。
本实施例涉及的主要实验部分如下：
1、RIP-PCR检测LARP6结合胶原mRNA水平
方法同实施例2中RIP-PCR检测，结果见图7A-C。
2、RT-PCR检测Bumetanide对胶原mRNA产生的影响
方法同实施例1中RT-PCR检测，结果见图8A-C。
小结：以上结果表明Bumetanide在人源的成纤维细胞中也有抑制胶原产生的功能。
综合上述各实施例，本发明从天然活性化合物库出发，经过计算机模拟筛选，在细胞动物水平反复研究，发现在C188-9，Venetoclax和Bumetanide可参考作为临床上治疗纤维化疾病的一个新的潜在药物。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。

Claims (6)

  1. C188-9、Venetoclax、Bumetanide中的至少一化合物作为预防和治疗纤维化疾病药物中的应用；
    其中，C188-9(式I)、Venetoclax(式Ⅱ)、Bumetanide(式Ⅲ)的结构式分别为：
    Figure PCTCN2021076760-appb-100001
  2. 根据权利要求1所述应用，其特征在于，所述功能产品为能够对机体器官纤维化疾病的发生、发展产生治疗、缓解、抑制、调节的有益作用的产品或潜在物质；所述功能产品为单一制剂或包含有效量制剂成分的组合物。
  3. 根据权利要求1所述应用，其特征在于，所述功能产品以LARP6蛋白的RRM区域为靶标。
  4. 根据权利要求1所述应用，其特征在于，所述功能产品用于起到以下一种或多种作用：
    i)降低成纤维细胞中的胶原的表达；
    ii)抑制成纤维细胞中的LARP6与胶原mRNA的结合。
  5. 根据权利要求1所述应用，其特征在于，所述纤维化疾病包括肺纤维化、肝纤维化、肾纤维化、心纤维化、子宫内膜纤维化、眼纤维化、胰腺纤维化、脾纤维增生疾病、骨髓纤维化疾病或由纤维化诱发的疾病。
  6. 如权利要求5所述用途，其特征在于，所述肺纤维化疾病是药物性肺纤维化，药物性肺纤维化是结节病、尘肺、过敏性肺炎或放射线诱发的肺纤维化，其他病因未明的肺纤维化，以及由肺纤维化诱发的疾病；所述肝纤维化是病毒性肝炎、酒精性肝炎、自身免疫性疾病、脂肪肝、营养不良、慢性充血性心力衰竭或药物引起的肝脏纤维化，其他病因未明的肝脏纤维化，以及由肝纤维化诱发的疾病；所述肾纤维化是高血压、肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥、肾盂肾炎、肾结石、高血脂、糖尿病、高尿酸尿症、高钙尿症 引起的肾脏纤维化，其他病因未明的肾脏纤维化，以及由肾纤维化诱发的疾病；所述心纤维化是缺血性心脏疾病、高血压、病毒性心肌炎、代谢性心肌病、克山病、扩张性心肌病、肥厚性心肌病、限制性心肌病或心律失常引起的心脏纤维化、心脏重构和心肌肥厚，其他病因未明的心纤维化，以及由心纤维化诱发的疾病；所述子宫内膜纤维化是不同原因引起的子宫内膜纤维化病变，以及由子宫内膜纤维化诱发的疾病；所述眼纤维化是眼睛外伤、眼睛手术或糖尿病引起的眼视网膜纤维增生疾病，以及由眼纤维化诱发的疾病。
PCT/CN2021/076760 2020-08-03 2021-02-18 C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用 WO2022027953A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010767678.8A CN111973578A (zh) 2020-08-03 2020-08-03 C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用
CN202010767678.8 2020-08-03

Publications (1)

Publication Number Publication Date
WO2022027953A1 true WO2022027953A1 (zh) 2022-02-10

Family

ID=73445768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/076760 WO2022027953A1 (zh) 2020-08-03 2021-02-18 C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用

Country Status (2)

Country Link
CN (1) CN111973578A (zh)
WO (1) WO2022027953A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111973578A (zh) * 2020-08-03 2020-11-24 天津医科大学 C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用
CN113907047B (zh) * 2021-12-13 2022-03-11 天津医科大学总医院空港医院 一种自身抗原表位诱导的eam小鼠模型的建立方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010062640A1 (en) * 2008-10-28 2010-06-03 Gilead Colorado, Inc. Methods for treating idiopathic pulmonary fibrosis and associated complications
WO2015010102A1 (en) * 2013-07-18 2015-01-22 Baylor College Of Medicine Methods and compositions for treatment of fibrosis
WO2015010107A1 (en) * 2013-07-18 2015-01-22 Baylor College Of Medicine Methods and compositions for treatment of muscle wasting, muscle weakness, and/or cachexia
CN105377289A (zh) * 2013-04-21 2016-03-02 耶达研究及发展有限公司 用于下调Bcl-xL和/或Bcl-w的活性和/或量的试剂
CN106163557A (zh) * 2014-01-28 2016-11-23 巴克老龄化研究所 用于杀死衰老细胞和用于治疗衰老相关疾病和病症的方法和组合物
CN111973578A (zh) * 2020-08-03 2020-11-24 天津医科大学 C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010062640A1 (en) * 2008-10-28 2010-06-03 Gilead Colorado, Inc. Methods for treating idiopathic pulmonary fibrosis and associated complications
CN105377289A (zh) * 2013-04-21 2016-03-02 耶达研究及发展有限公司 用于下调Bcl-xL和/或Bcl-w的活性和/或量的试剂
WO2015010102A1 (en) * 2013-07-18 2015-01-22 Baylor College Of Medicine Methods and compositions for treatment of fibrosis
WO2015010107A1 (en) * 2013-07-18 2015-01-22 Baylor College Of Medicine Methods and compositions for treatment of muscle wasting, muscle weakness, and/or cachexia
CN106163557A (zh) * 2014-01-28 2016-11-23 巴克老龄化研究所 用于杀死衰老细胞和用于治疗衰老相关疾病和病症的方法和组合物
CN111973578A (zh) * 2020-08-03 2020-11-24 天津医科大学 C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
LI ZILONG, CHEN BAOYU, DONG WENHUI, KONG MING, FAN ZHIWEN, YU LIMING, WU DONGMEI, LU JUN, XU YONG: "MKL1 promotes endothelial-to-mesenchymal transition and liver fibrosis by activating TWIST1 transcription", CELL DEATH & DISEASE, NATURE PUBLISHING GROUP, GB, vol. 10, no. 12, 1 December 2019 (2019-12-01), GB , pages 899 - 13, XP055895102, ISSN: 2041-4889, DOI: 10.1038/s41419-019-2101-4 *
PEDROZA MESIAS, TO SARAH, ASSASSI SHERVIN, WU MINGHUA, TWEARDY DAVID, AGARWAL SANDEEP K: "Role of STAT3 in skin fibrosis and transforming growth factor beta signalling", RHEUMATOLOGY, OXFORD UNIVERSITY PRESS, LONDON, GB, vol. 57, no. 10, 1 October 2018 (2018-10-01), GB , pages 1838 - 1850, XP055895142, ISSN: 1462-0324, DOI: 10.1093/rheumatology/kex347 *
SAMANANI SHAZIA; MISHRA MANOJ; SILVA CLAUDIA; VERHAEGHE BROOKE; WANG JANET; TONG JADE; YONG V WEE: "Screening for Inhibitors of Microglia to Reduce Neuroinflammation", CNS & NEUROLOGICAL DISORDERS, BENTHAM SCIENCE PUBLISHERS LTD.,, NL, vol. 12, no. 6, 1 September 2013 (2013-09-01), NL , pages 741 - 749, XP009194559, ISSN: 1871-5273, DOI: 10.2174/18715273113126660177 *
SHUANG LIU, YISHENG LIU, SIQI YING, LINRONG PAN, GENGRU JIANG: "Function of NKCC1 in the Aortic Fibrosis of Rats with Chronic Kidney Disease", CHINESE JOURNAL OF CELL BIOLOGY, ZHONGGUO KEXUEYUAN SHANGHAI SHENGMING KEXUE YANJIUYUAN SHENGWU HUAXUE YU XIBAO SHENGWUXUE YANJIUSUO, CN, vol. 42, no. 5, 1 January 2020 (2020-01-01), CN , pages 763 - 771, XP055895141, ISSN: 1674-7666, DOI: 10.11844/cjcb.2020.05.0003 *
TENG KUN-YU, BARAJAS JUAN M., HU PENG, JACOB SAMSON T., GHOSHAL KALPANA: "Role of B Cell Lymphoma 2 in the Regulation of Liver Fibrosis in miR-122 Knockout Mice", BIOLOGY, MDPI, CH, vol. 9, no. 7, 1 July 2020 (2020-07-01), CH , pages 157 - 18, XP055895113, ISSN: 2079-7737, DOI: 10.3390/biology9070157 *
VAN ANDEL DORINDE M., SPRENGERS JAN J., ORANJE BOB, SCHEEPERS FLOORTJE E., JANSEN FLOOR E., BRUINING HILGO: "Effects of bumetanide on neurodevelopmental impairments in patients with tuberous sclerosis complex: an open-label pilot study", MOLECULAR AUTISM, BIOMED CENTRAL LTD, LONDON, UK, vol. 11, no. 1, 1 December 2020 (2020-12-01), London, UK , pages 30 - 14, XP055895117, ISSN: 2040-2392, DOI: 10.1186/s13229-020-00335-4 *

Also Published As

Publication number Publication date
CN111973578A (zh) 2020-11-24

Similar Documents

Publication Publication Date Title
WO2022027953A1 (zh) C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用
Ge et al. MFGE8 attenuates Ang-II-induced atrial fibrosis and vulnerability to atrial fibrillation through inhibition of TGF-β1/Smad2/3 pathway
CN101300346B (zh) 新型生理物质nesfatin及其相关物质、以及它们的用途
WO2012006577A2 (en) Direct reprogramming of cells to cardiac myocyte fate
BR112012031587B1 (pt) Composição compreendendo um peptídeo e pelo menos um outro componente farmaceuticamente aceitável para o tratamento ou prevenção de dano de cartilagem e artrite
US8404644B2 (en) Agents with angiogenic and wound healing activity
Shao et al. Cell and molecular regulation of endothelin-1 production during hepatic wound healing
Chen et al. Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells
US10815312B2 (en) Bifunctional peptide having capability to permeate cells and capability to regenerate muscles and use thereof
Liang et al. Effects of miR-145-5p on cardiomyocyte proliferation and apoptosis, GIGYF1 expression and oxidative stress response in rats with myocardial ischemia-reperfusion
WO2022100720A1 (zh) 一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病
RU2378375C2 (ru) Способ пролиферации кардиомиоцитов
Piscaglia et al. Expression of ECM proteins fibulin-1 and-2 in acute and chronic liver disease and in cultured rat liver cells
Igoucheva et al. Protein therapeutics for Junctional Epidermolysis bullosa: incorporation of recombinant β3 chain into laminin 332 in β3-/-keratinocytes in vitro
CN114790234B (zh) 一种脂肪干细胞分泌型内源性多肽adscp5及其应用
JP4406013B2 (ja) 細胞の接着・伸展を促進するペプチド、その断片及びその誘導体
EP2619330B1 (en) Method for identifying compounds to increase the expression or activity of aspartyl aminopeptidase
WO2003053467A1 (fr) Medicament pour regenerer les tissus et les vaisseaux et procede permettant de le produire
Zhang et al. Novel Pro-myogenic Factor Neoruscogenin Induces Muscle Fiber Hypertrophy by Inhibiting MSTN Maturation and Activating the Akt/mTOR Pathway
KR20080044205A (ko) 저산소 스트레스 응답에 관여하는 Int6 단백질, 및 그 용도
Qubisi Glomerulus injury and repair in the zebrafish (Danio rerio)
Bronstein et al. cDNA cloning and spatiotemporal expression during avian embryogenesis of hnRNP A1, a regulatory factor in alternative splicing
Commons Understanding The Functions Of Vimentin Filaments In Collagen Expression And Targeting Vimentin Filaments For The Treatment Of Fibrosis
Phyo Regulation of the Tubulin Code in Cardiac Hypertrophy and Failure
Onursal Differential effects of prolyl-3-hydroxylases 1 and 4 on collagen biosynthesis highlight prolyl-3-hydroxylase 4 as a potential novel drug target in pulmonary fibrosis

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: 21852680

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21852680

Country of ref document: EP

Kind code of ref document: A1