WO2016192523A1 - Utilisation d'artépilline c et d'analogues de ceux-ci dans la préparation de médicaments pour la prévention et le traitement de maladies métaboliques - Google Patents

Utilisation d'artépilline c et d'analogues de ceux-ci dans la préparation de médicaments pour la prévention et le traitement de maladies métaboliques Download PDF

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WO2016192523A1
WO2016192523A1 PCT/CN2016/081956 CN2016081956W WO2016192523A1 WO 2016192523 A1 WO2016192523 A1 WO 2016192523A1 CN 2016081956 W CN2016081956 W CN 2016081956W WO 2016192523 A1 WO2016192523 A1 WO 2016192523A1
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artepillin
creb
crtc2
liver
disease
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刘浥
陈亚琼
周瓒
袁源
孙秀杰
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid

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  • the present invention belongs to the field of biomedicine, and more particularly, the present invention relates to the use of atropin C and analogs thereof for the preparation of a medicament for the prevention and treatment of metabolic diseases.
  • the liver is central to the regulation of blood glucose processing, including absorption of blood sugar, glycogen conversion, and gluconeogenesis and output.
  • Hepatic gluconeogenesis is an important pathology in the increase of hepatic glucose output in type 2 diabetes. Correcting hyperglycemia of hepatic glucose is beneficial to control the progression of diabetes and the secondary disease. Therefore, studies of inhibitors targeting hepatic glucose production are important for the control and treatment of metabolic syndrome, including hyperglycemia, obesity, and type 2 diabetes.
  • Glucagon receptor is activated during starvation, Glucagon receptor (GCGR) is activated, and 3,5-cyclic adenosine monophosphate (cAMP) is synthesized by adenylate cyclase on G ⁇ s-activated membrane.
  • CREB CRE response element-binding protein
  • CRTCs from Drosophila, C. elegans to humans: including three members, CRTC1, CRTC2 and CRTC3. They have similar protein molecular structures (CRTC2 as an example, Figure 11), both containing the N-terminal CREB binding domain (CBD, CREB binding domain), central regulatory region (REG, regulatory domain), shear region (SD, splicing domain) And the C-terminal transcriptionally active region TAD (transcription active domain).
  • CBD N-terminal CREB binding domain
  • REG central regulatory region
  • SD shear region
  • TAD transcription active domain
  • the N-terminal CBD forms a helix-helical structure that binds to the ZIP region of CREB and is a characteristic structure of the CRTCs family.
  • the NLS (nuclear leading signaling) region and the nuclear export signal region (NES) are located in the central regulatory region REG, of which 171 is serine. Phosphorylation regulates the nuclear shuttling process of CRTC2.
  • the C-terminal TAD (transcription active domain) has transcriptional activity in combination with TAF.
  • the three subtypes of CRTC have characteristic distribution.
  • CRTC1 is mainly expressed in brain tissue.
  • CRTC2 is the highest expressed in liver tissue.
  • the high abundance expression of CRTC3 is lymphocytes and lungs. It is digested in muscle, liver, pancreas and intestine. The expression level in the system is low.
  • CRTC2 is an important transcriptional regulatory coactivator in the hepatic gluconeogenesis pathway ( Figure 12). Under resting conditions, S171 of CRTC2 is continuously phosphorylated by SIK2 (Salk induced kinases 2), and is retained in the cytoplasm by binding to cytoplasmic protein 14-3-3.
  • SIK2 Salk induced kinases 2
  • Liver CRTC2 integrates hormones, trophic factors (cAMP agonists, glucagon, insulin) and various regulatory proteins (CRY1, AMPK, SIK, SMEK1/2) to regulate transcription of hepatic gluconeogenesis.
  • cAMP agonists cAMP agonists
  • glucagon glucagon
  • regulatory proteins CRY1, AMPK, SIK, SMEK1/2
  • CRTC2 also mediates the hypoglycemic effects of anti-diabetic first-line clinical drugs metformin and thiazolidinedione, which indirectly enhance CRTC2 phosphorylation and inhibit glycocalyx in type 2 diabetic patients by regulating the kinase activity of AMPK.
  • metformin and thiazolidinedione which indirectly enhance CRTC2 phosphorylation and inhibit glycocalyx in type 2 diabetic patients by regulating the kinase activity of AMPK.
  • the resulting improvement in hyperglycemia Therefore, compounds
  • Glucagon receptor Glucagon receptor
  • FBPase fructose-1, 6-biphosphatase
  • G6Pase glucose-6-phosphatase.
  • glucagon receptor small molecule inhibitors such as NNC92-1687, Bay-27-9955, and trisubstituted urea
  • glucagon receptor high potency monoclonal antibody have good Prospects for hypoglycemic applications.
  • these inhibitors are all based on the function of inhibiting the target, and can not improve the pathology of such target expression.
  • the liver is also an important organ for the metabolism of lipids (fatty acids, cholesterol and phospholipids).
  • the liver can break down triglycerides into fatty acids and secrete them into the circulatory system for use by other tissues.
  • the liver also synthesizes a large amount of lipoprotein to assist in the secretion of fat.
  • excess carbohydrates and proteins are present, the liver converts excess carbohydrates and proteins into fatty acids and triglycerides for transfer to adipose tissue for storage.
  • the metabolic process of synthesis, absorption and conversion of cholesterol into cholic acid is also performed by the liver.
  • Atropin C or an analogue thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for preventing, ameliorating or treating a metabolic disease.
  • the metabolic disease comprises: a lipid metabolic disease, a glucose metabolism disease, or a metabolic syndrome.
  • the lipid metabolic diseases include, but are not limited to, hyperlipemia, fatty liver, obesity, atherosclerosis, coronary heart disease, hypertension, cerebral infarction, stroke, renal failure.
  • the glucose metabolism diseases include, but are not limited to, hyperglycemia, diabetes, obesity, hyperinsulinemia, atherosclerosis, coronary heart disease, hypertension, hyperthyroidism, diabetic eye disease , diabetic nephropathy.
  • the atropin C or analog thereof has the structure of the following formula (I):
  • R1 is selected from the group consisting of H and prenyl
  • R2 is OH
  • R3 is selected from the group consisting of H, isopentenyl
  • R4 is selected from H, CH 2 Ph, Or CH 2 CHPh.
  • the compound to which it belongs is selected from the group consisting of: atropin C;
  • R1 is H
  • R2 is OH
  • R3 is isopentenyl
  • R4 is H
  • R1 is H, R2 is OH, R3 is H, R4 of compound is CH 2 Ph; or
  • R1 is H
  • R2 is OH
  • R3 is H
  • R4 is compound of.
  • the medicament is further used to:
  • a method of preparing a medicament for preventing, ameliorating or treating a medicament for a lipid metabolism disease or a glucose metabolism disease comprising: administering an effective amount of atropin C or an analog thereof or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable carrier.
  • the metabolic disease comprises: a lipid metabolic disease, a glucose metabolism disease, or a metabolic syndrome.
  • the atropin C or analog thereof has the structure of the following formula (I):
  • R1 is selected from the group consisting of H and prenyl
  • R2 is OH
  • R3 is selected from the group consisting of H, isopentenyl
  • R4 is selected from H, CH 2 Ph, Or CH 2 CHPh.
  • GTT Glucose tolerance
  • ITT Insulin sensitivity test of db/db mice after oral administration of Brazilian green propolis for 2 weeks;
  • E and DIO mice were treated with Brazilian green propolis for 3 weeks, and QPCR was used to detect mRNA transcription levels of hepatic gluconeogenesis key genes.
  • G Analysis of the transcriptional activity of the reporter gene G6P-Luc in primary hepatocytes.
  • the primary cells of the liver were pretreated with propolis (0.2%) for 1 hour and then stimulated by glucagon (100 nM) for 6 hours;
  • CREB-CRTC2 mammalian two-hybrid system was established, expression plasmids AD-hCRTC2-Ser171Ala, BD-hCREB, Gal4-Luc and RSV-Renilla were transferred into HEK293T for transient expression;
  • CREB-CRTC2 two-hybrid system was used to evaluate the biological activity of the Brazilian green propolis monomer, and the test compound (25 ⁇ M) was added for 4 hours after transfection for 6 hours;
  • Artepillin C inhibits the binding of endogenous CREB to FLAG-mCRTC2 in hepatic primary cells.
  • Artepillin C (10 ⁇ M) pretreated the primary liver cells overexpressing HA-CRTC2 for 1 hour, pancreas Glucagon was stimulated for 30 minutes, and co-immunoprecipitation system including anti-CREB as primary antibody, including Artepillin C (10 ⁇ M), was incubated overnight;
  • GST-pull down was used to detect the inhibitory activity of Artepillin C on the binding of purified protein GST-CREB to HIS-CRTC2-Ser171Ala.
  • GST-CREB beads immunoprecipitated and purified protein HIS-CRTC2-Ser171Ala, and the co-immunoprecipitation system contained Apripillin C ( 0,5,10,50 ⁇ M);
  • SPR Surface plasmon resonance
  • GST-CREB and N' truncated bodies including GST-CREB341 full length, GST-CREB ⁇ Q1 (102-341), GST-CREB ⁇ Q1 ⁇ KID (160-341), GST-CREB-bZIP (283-341), GST antibody detection;
  • Artepillin C inhibits the binding curve of CREB-CRTC2 binding, and the IC50 of Artepillin C is detected by mammalian CREB-CRTC2 two-hybrid system.
  • CHIP-QPCR was used to detect the effect of Artepillin C on the binding of CRTC2 and CREB to CRE elements induced by glucagon.
  • the primary hepatocytes after infection with adenovirus Ad-HA-CRTC2 were pretreated with Artepillin C (10 ⁇ M) for 1 hour.
  • CHIP-QPCR assay was performed after stimulation of glucagon (100 nM) for 30 minutes;
  • CRTC2-deficient primary hepatocytes (CRTC2 -/- ) glucose glucose output detection, Artepillin C (10 ⁇ M) pretreatment of CRTC2-deficient primary hepatocytes (CRTC2 -/- ) for 1 hour, glucagon (100 nM) ) stimulation for 4 hours, sugar production reaction for 8 hours;
  • DIO mice left, 0, 10, 20 mg/kg
  • db/db model mice right, 20 mg/kg
  • Artepillin C 5 weeks
  • Artepillin C inhibits weight gain in diabetic model mice (left, DIO, right, db/db);
  • TC total cholesterol
  • LDLC low density cholesterol
  • HDLC high density cholesterol
  • DIO mice were injected intraperitoneally with Artepillin C (20mg/kg) for 5 weeks, QPCR detection of DIO mice Transcription of hepatic gluconeogenesis genes in liver tissue;
  • DIO mice starved blood glucose monitoring, high-fat induced diabetic rats (DIO) intraperitoneal injection of Artepillin C (20mg / kg) 1 week, 16 hours of starvation blood glucose levels;
  • mice were injected intraperitoneally with Artepillin C (20 mg / kg) for 5 weeks, serum insulin levels were tested;
  • Figure 9 Molecular mechanism by which Artepillin C increases overall insulin sensitivity.
  • APC analogs inhibit the activity of CREB/CRTC2 binding to each other;
  • the inventors have extensively and intensively studied, for the first time, to reveal that Artepillin C (APC) or an analogue thereof has an extremely remarkable effect on the prevention and treatment of metabolic diseases.
  • the present invention provides a multifunctional drug that targets CREB/CRTC2 and SREBP.
  • the present invention provides Artepillin C (APC) or an analog thereof having the chemical structure of the following formula (I):
  • the compound of the structure of the formula (I) forms a suitable compound spatial configuration, and the compound having the structure can target the CREB/CRTC2 pathway and interact with the CREB-related site to regulate the downstream one.
  • the series of mechanisms of action That is, the atropin C or analog thereof is an inhibitor of the CREB/CRTC2 pathway.
  • the compound is attropin C and has the structural formula:
  • the present invention also includes pharmaceutically acceptable salts, hydrates or precursors of the above compounds as long as they also have an effect of preventing, alleviating or treating metabolic diseases.
  • pharmaceutically acceptable salt refers to a salt formed by reacting a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal.
  • These salts include, but are not limited to, (1) salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid , methanesulfonic acid, maleic acid, or arginine.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, in the form of esters, carbamates, or other conventional "prodrugs".
  • precursor of a compound refers to a compound, or a structural formula, which is converted into a structure of the formula (I) by a metabolic or chemical reaction of a precursor of the compound in a patient after administration by an appropriate method.
  • precursor of the compound in a patient after administration by an appropriate method.
  • the present invention also includes isomers and racemates of the above compounds as long as they also have an effect of preventing metabolic diseases.
  • the term "isomer” as used herein includes: geometric isomers, enantiomers, non-pairs Enantiomers (eg, cis-trans isomers, conformational isomers).
  • the compound has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.
  • the compounds of the present invention can be obtained by a variety of methods well known in the art, using known starting materials, such as chemical synthesis or from organisms (such as animals or plants). The method of extraction, which is included in the present invention.
  • Some of the compounds having the structure of formula (I) can also be extracted, isolated and purified from organisms such as animals or plants.
  • the present inventors have found that atropin C inhibits the binding of CRTC2 and CREB after nuclear entry by directly binding to the N-terminus of CREB, and reduces the expression of downstream genes driven by CREB/CRTC2, including hepatic gluconeogenesis.
  • APC inhibits the expression of SREBPs driven by LXRE, inhibits the expression of SREBPs driven by LXRE, inhibits the downstream target genes of SREBPs, and lowers the cholesterol and fatty acids regulated by SREBPs by inhibiting the expression of nuclear receptor LXR ⁇ and synergizing the transcriptional coactivator PGC1 ⁇ . The synthesis, absorption, and ultimately reduce the fat content of the blood.
  • the present invention provides the use of a compound having the structure of the formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or a precursor thereof, for use in A medicament (or composition) for controlling a metabolic disease is prepared.
  • the metabolic disease includes a lipid metabolism disease or a glucose metabolism disease.
  • the lipid metabolic diseases include: hyperlipemia, fatty liver, obesity, atherosclerosis, coronary heart disease, hypertension, cerebral infarction, stroke, renal failure and the like.
  • the diseases of glucose metabolism include: hyperglycemia, Diabetes, obesity, hyperinsulinemia, atherosclerosis, coronary heart disease, hypertension, hyperthyroidism, diabetic eye disease, etc.
  • the compounds of the present invention regulate lipid metabolism and glucose metabolism through a novel mechanism, and have been shown to have significant effects on lipid metabolism diseases or glucose metabolism diseases through animal experiments.
  • composition of the invention is generally a pharmaceutical composition comprising attilin C or an analog thereof or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate thereof. Or a precursor as an active ingredient for the prevention and treatment of metabolic diseases; and a pharmaceutically acceptable carrier or excipient.
  • the term "containing” means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • a "pharmaceutically acceptable" ingredient is a substance which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxicity, irritation, and allergy), i.e., has a reasonable benefit/risk ratio.
  • a "pharmaceutically acceptable carrier” is used to use the present invention, aspirin C or an analog thereof, or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or The pharmaceutically or foodly acceptable solvent, suspending agent or excipient that the precursor delivers to the animal or human.
  • the carrier can be a liquid or a solid.
  • Pharmaceutically acceptable carriers suitable for use in the present invention include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the present invention also provides a method of preparing a composition for preventing and treating a metabolic disease, which comprises using an effective amount of atpirin C or an analog thereof or an isomer thereof, a racemate, a pharmaceutically acceptable salt thereof.
  • the hydrate or precursor is mixed with a pharmaceutically acceptable carrier to obtain a composition of the present invention, and the proportion by weight of the active ingredient in the composition may be, for example, 0.0001 to 50% by weight; preferably 0.001 to 20% by weight.
  • the dosage form of the pharmaceutical composition of the present invention may be various, as long as it is a dosage form capable of effectively bringing the active ingredient to the affected part of the mammal.
  • the preferred pharmaceutical composition is an oral or injectable preparation.
  • it may be selected from the group consisting of granules, tablets, capsules, solutions, or suspensions, and powders.
  • Various conventional carriers or excipients such as fillers, flavoring agents, antioxidants, perfumes, pigments, lubricants, glidants, wetting agents, emulsifiers, which are required for the preparation of different dosage forms, may be added to the compositions of the present invention. , pH buffer substances, etc. These additives are well known to those skilled in the art.
  • the invention also provides a method for controlling a metabolic disease comprising the steps of: administering to a subject in need thereof an effective amount of atpirin C or an analog thereof or an isomer thereof, a racemate, pharmaceutically acceptable Salt, hydrate or precursor.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • a safe and effective amount of the compound will generally be from about 0.1 ng to about 100 mg/kg body weight; preferably from about 1 ng to 10 mg/kg body weight.
  • specific dosages should also take into account factors such as the route of administration, the health of the user, and the like, which are within the skill of the skilled physician.
  • the compounds of the invention may also be used with other active ingredients or therapeutic agents (such as other hypolipidemic drugs, cholesterol lowering drugs, diabetes drugs, etc.).
  • the ethyl acetate-40% fraction (3.2g) was separated by normal phase column chromatography and reverse phase ODS column; B-60% (21.8g) was subjected to repeated normal phase column, ODS column (methanol-water system), Sephadex LH- 20 gel column (methanol-water system elution) and other separation means separation; ethyl acetate - 80% of the part (37.2g) after normal phase column, recrystallization, ODS column (methanol-water system), Sephadex LH-20 condensation Separation by a gel column (methanol-water system elution), etc.; ethyl acetate-100% fraction (29.6 g) was subjected to normal phase column chromatography and recrystallization; the aqueous layer was separated by an ODS column. A monomeric compound of Brazilian green propolis was obtained.
  • CREB-CRTC2 mammalian two-hybrid system including human CREB and BD (Gal4-Binding domain), human CRTC2 and AD (Gal4-Active domain) fusion expression vector, combined with inducible Gal4-Luciferase and constitutive RSV- Renilla double reporter gene expression vector to create a complete CREB/CRTC2 mammalian two-hybrid system.
  • the fusion protein expression vector in the mammalian two-hybrid system was framed by plasmids 804 and 811 (obtained from Tongji University). Cloning sites and primers are shown in Table 1.
  • the two-hybrid reporter gene GAL4-Luciferase was purchased from (Promega) and the reference reporter gene RSV-Renilla was constructed as shown in Table 1.
  • the RSV promoter sequence was substituted for CMV-Renilla. (NEB) CMV promoter sequence.
  • the fusion protein HIS-CRTC2-S171A expression vector backbone is pET28C (purchased from Addgene), and the GST-tagged CREB full-length and N-terminal truncated fusion protein prokaryotic expression vector is pGEX5X-1 (GE) as the backbone, and the specific cloning position Point and PCR amplification primers are shown in Table 1.
  • HEK293T cells were grown in a logarithmic growth phase and cultured in 24-well culture plates overnight.
  • Liposome Lipo 2000, Invitrogen
  • the test compound and solvent control were added.
  • % DMSO 3 replicates per compound, the expression of the reporter gene was detected after 18 hours of culture, the expression level of the corrected Gal4-luciferase was evaluated, and the test compound was analyzed by the inhibition screening model.
  • the high-purity adenovirus was transfected into the short-term cultured primary hepatocytes into the inducible Ad-CRE-Luciferase and the constitutively expressed Ad-RSV-Renilla reporter gene to construct a double reporter gene detection system with glucagon
  • the test compound was used as an inhibitor to evaluate the inhibitory activity of the compound against the activity of the CRE-luciferase reporter gene.
  • a recombinant adenovirus expressing an inducible Ad-CRE-Luciferase was obtained from Dr. Marc R. Montminy, Salk Institute, USA. Construction of a recombinant adenoviral vector expressing a constitutively expressed Ad-RSV-Renilla reporter gene: The promoter CMV of the CMV-Renilla expression vector was replaced by a promoter RSV by cloning, and the fragment RSV-Renilla was cloned by subcloning.
  • the adenovirus shuttle vector pshutter, positive pshutter-RSV-Renilla was recombined with plasmid Ad-easy to obtain the Ad-easy-RSV-Renilla plasmid.
  • the positive recombinant plasmid Ad-easy-RSV-Renilla was digested with the endonuclease PacI to linearize Ad-easy-RSV-Renilla, and then transferred to the adenovirus packaging cell line MGH293 (obtained from Dr. Marc R. Montminy, Salk Institute, USA) ), plaques appear after 7-14 days to indicate successful packaging of adenovirus.
  • the adenovirus was amplified and purified by cesium chloride for use in mouse and cell experiments.
  • 25g or so wild-type C57 mice were fixed after anesthesia, and were perfused through the portal-inferior vena cava.
  • the pre-HBSS perfusion solution cleared the blood in the liver.
  • Collagenase (Sigma) digested extracellular collagen to separate cells, 100 mesh. After the cell sieve was used to filter the tissue fragments, the primary liver cells were washed twice with serum-free M199 medium, and the treated liver primary cells were cultured in serum-containing M199 medium. Starved for 16 hours in serum-free M199 medium before the experiment.
  • hepatocytes Primary hepatocytes were seeded in 96-well cell plates (3000 cells/well), starved for 16 hours in serum-free M199, and tested at different concentrations for 1-2 days. After removing the medium, add 20 ul of MTT reagent. (5 ⁇ g/ml, pH 7.4) was co-cultured with the cells for 5 hours, and MTT was sufficiently dissolved after adding 150 ⁇ l of DMSO, and the absorbance of OD490 was examined to evaluate the effect of the test compound on the vitality of primary hepatocytes.
  • MTT reagent 5 ⁇ g/ml, pH 7.4
  • the treated cells or cryopreserved tissues were lysed in RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6), and fully lysed. After centrifugation, the supernatant was taken. After moderate dilution, the total protein concentration of the sample was determined by BCA method. The sample with the same amount of total protein was added to 5 ⁇ SDS and denatured at 95 °C for 5 minutes to separate different proteins with a certain concentration of SDS-PAGE gel. And then in the electric field to the PVDF membrane. The antibody is used to detect changes in the phosphorylation or expression level of the target protein in the total protein.
  • the treated cells or cryopreserved tissues were lysed in IP buffer, fully lysed, and then centrifuged to remove the supernatant. After moderate dilution, the total protein concentration of the sample was determined by BCA method, and 10% of the lysate was taken as the Input group. A lysate containing 1 ⁇ g of total protein was added, and Protein A/G beads (Millipore) was added and the primary antibody of the target protein was identified, and then incubated at 4 °C. The precipitated beads were washed, and the protein complex bound to the antibody immunoprotein was eluted by adding 3 ⁇ SDS buffer at 95 ° C for 6 minutes. Western blot analysis of changes in target proteins in immunoprecipitated samples.
  • the FLAG-mCRTC2 plasmid was obtained from Dr. Marc R. Montminy, Salk Institute, USA.
  • HIS-tag fusion human CRTC2-Ser171Ala recombinant protein prokaryotic expression vector (Table 1): Human CRTC2-Ser171Ala expression sequence was cloned into the prokaryotic expression vector pET28C by EcoRI/HindIII, and CRTC2-Ser171Ala was fused and integrated with HIS by sequencing. The construction of the recombinant protein prokaryotic expression vector of the GST-tagged fusion CREB is shown in Table 1. Human CREB (hCREB) was cloned into the prokaryotic expression vector pGEX5X1 by EcoRI/NotI, and sequenced and fused to the tag GST and fully expressed.
  • the prokaryotic expression vector was transferred into E. coli and induced to express by IPTG at 16 ° C overnight. After collecting the cells, the cells were lysed, sonicated, and the supernatant was taken after centrifugation, and the tagged protein was purified by GST beads or HIS beads.
  • the lysate supernatant of the GST-fused CREB recombinant protein was incubated with GST beads for 30 minutes at room temperature, and the non-specific binding protein on the beads was washed to remove GST-CREB beads.
  • HIS-tag fusion human CRTC2-Ser171Ala recombinant protein (HIS-CRTC2-Ser171Ala) was induced to express in bacteria, and the HIS-CRTC2-Ser171Ala protein was purified by HIS beads, and the imidazole was removed by desalting. The obtained purified protein HIS-CRTC2-Ser171Ala was used for Pull down.
  • HIS-CRTC2-Ser171Ala purified protein 800 ng of HIS-CRTC2-Ser171Ala purified protein, 40 ⁇ l of GST-CREB beads and test compound were included in a 500 ⁇ l pull down system and incubated at 4 °C. The bound beads were washed, the non-specific binding protein was removed, and the bound protein sample was obtained by adding 3X SDS buffer at 95 ° C for 6 minutes. Western blot analysis of protein changes in the eluted samples.
  • hepatocytes Primary cultured hepatocytes were used as experimental materials, pretreated with test compound for 1 hour, then 100 nM glucagon was stimulated for 30 minutes, and the supernatant was removed. After washing with PBS, the cells were lysed according to the cAMP ELISA (R&D) kit. Description of the operation, evaluation of test compound on glucagon-induced cAMP The impact of the formation.
  • the primary liver cells were hungry overnight, and the test compound was pre-incubated for 1 hour. After stimulation with 100 nM glucagon for 4 hours, the medium was replaced with sugar-free, sodium pyruvate-free, phenol-free medium (Sigma). Incubate with sodium pyruvate and sodium lactate for 8 hours, collect the medium and centrifuge, and determine the glucose content in the medium according to the kit (GO kit, Sigma) to evaluate the ability of the test compound to produce sugar to the primary hepatocytes. influences.
  • the cultured cells or frozen tissues were lysed in Trizol, and total RNA was extracted by isopropanol precipitation, washed with 80% ethanol (DEPC), dried, and dissolved in EDPC water.
  • the cDNA (TAKARA) was reverse transcribed using 0.5-1 ⁇ g of total RNA as a template.
  • the target gene IBA7900 was amplified by QPCR method, and the change in mRNA copy number of each target gene was analyzed by detecting the Ct value.
  • SPR Surface plasmon resonance
  • the channel for capturing Ni but not binding protein is set as the reference channel during the detection.
  • the RU value on the experimental channel is deducted from the reference channel RU value and is the original data.
  • the mobile phase molecular concentration and its response RU value are fitted to the binding curve by a single site pattern to obtain the kinetic parameters of the pair of molecules when they are combined with each other.
  • HIS-tag fusion of human CREB recombinant protein Human CREB-encoding gene was cloned into prokaryotic expression vector pET28C by homologous recombination, and CREB and HIS were fused and fully expressed. HIS-CREB was transferred into E. coli expression strain BL21, and after induction, the cells were collected, sonicated, and the supernatant of the lysate was separated. HIS-CREB was affinity-purified with HIS beads, and HIS-CREB eluted with high salt was removed by dialysis. Imidazole, used for affinity experiments with small molecules and proteins.
  • Isothermal titration calorimetry measures the direct binding between small molecules and proteins by monitoring the change in heat (exothermic or endothermic) produced by the interaction of components.
  • the purified protein HIS-CREB was desalted and concentrated, dissolved in PBS (8.0, NaCL 500 mM), and dropped into the small molecule dilution to be tested.
  • the amount of heat released by the system was determined after each titration.
  • the change in heat of the injection is plotted as the peak area of the heat change peak and the molar ratio of the injected molecules. Binding isotherms were fitted to the single site pattern by ORIGIN7 software to obtain binding constants, stoichiometry, entropy and enthalpy parameters.
  • GTT Mouse glucose tolerance
  • PTT pyruvate tolerance
  • ITT insulin sensitivity
  • mice After the experimental mice were treated, they were starved overnight before the GTT experiment. Each mouse was intraperitoneally injected with sterile glucose (2 g/kg) according to the body weight, and blood was taken from the tail vein at 0, 15, 30, 60, and 120 minutes after the injection. The blood glucose concentration was measured by a blood glucose meter (Roche), and the blood glucose concentration curve of the mice in different treatment groups was made with time as the horizontal axis and blood glucose concentration as the ordinate. The PTT experimental injection was sodium pyruvate (1.5 g/kg). Mice were starved for 3 hours before the ITT experiment and the injection was insulin (1.5 U/kg).
  • the adenovirus carrying the reporter gene was injected into the tail vein, including the reporter gene expressing the inducible Ad-G6P-Luciferase reporter gene and the constitutive Ad-RSV- ⁇ -gal, and the in vivo imaging experiment was performed 3 days later.
  • the mice were starved overnight before the experiment, and the mice were anesthetized with isoflurane (Abbott), and the luciferase substrate Luciferin (Biosynth AG) was injected subcutaneously. After 15 minutes, the mouse liver luciferase was collected by an active imager (IVIS lumina, Xenogen).
  • the bioluminescence signal was analyzed for the ⁇ -gal value-corrected luciferase signal, and the effect of the test compound on the relative activity of G6P-Luciferase induced by starved glucagon in vivo was evaluated.
  • Ad-HA-mCRTC2 Ad-G6p-luc, Ad-CRE-Luc.Ad- ⁇ -Gal; all obtained from Dr. Marc R. Montminy of Salk Institute, USA.
  • a method for constructing a recombinant adenoviral vector expressing a constitutively expressed Ad-RSV-Renilla reporter gene the promoter CMV of the CMV-Renilla expression vector is replaced with a promoter RSV by cloning. After subcloning, the fragment RSV-Renilla was cloned into the adenoviral shuttle vector pshutter, and the positive pshutter-RSV-Renilla was recombined with the plasmid Ad-easy to obtain the Ad-easy-RSV-Renilla plasmid.
  • Ad-easy-RSV-Renilla was digested with the endonuclease PacI to linearize Ad-easy-RSV-Renilla, and then transferred to the adenovirus packaging cell line MGH293. After 7-14 days, plaques appeared to indicate adenoviral packaging. success.
  • the adenovirus was amplified and purified by cesium chloride for use in mouse and cell experiments.
  • the medium was collected, and the cells were collected by centrifugation and repeatedly frozen and thawed three times, and the virus particles were precipitated under saturated ammonium sulfate, and then the virus was resuspended in PBS.
  • the virus was purified twice in a CsCl density gradient and subsequently dialyzed against PBS-glycerol (10%) buffer overnight to remove the inorganic salts of the virus and stored at -80 °C.
  • the tissues were removed and embedded in paraffin.
  • the cells were observed by HE staining. Take a photo with the Olympus camera.
  • hepatocytes were cross-linked with formaldehyde and scraped in PBS.
  • the nuclei were collected in a lysis buffer, pulsed with 50% power for 1 second, with a gap of 2 seconds, and sheared for 3 minutes. The supernatant was used for subsequent experiments.
  • the cleaved chromosomal solution was incubated with IgG (Santa Cruz), CREB (Cell Signal) antibody and CRTC2 (Calbiochem) antibody at 4 ° C for 2H, respectively, followed by addition of Protein A beads (Milliopre) at 4 ° C overnight.
  • Immunoprecipitated chromatin was purified by Chelex 100 (Biored) and quantified by QPCR (ABI 7900) method.
  • the antibody CREB and CRTC2 immunoprecipitation-QPCR signals were corrected with the corresponding IgG signal as an internal standard.
  • the PCR primers used are listed in Table 2.
  • the supernatant obtained after centrifugation of the eyelid blood by EDTA anticoagulation treatment (3000 rpm, 15 minutes) was used to detect serum total triglyceride (TG), total cholesterol (TC, total cholesterol), low density cholesterol (LDLC, LDL).
  • TG serum total triglyceride
  • TC total cholesterol
  • LDLC low density cholesterol
  • the content of cholesterol and HDL cholesterol is in accordance with the kit (Beihua Kangtai).
  • the content of triglyceride and cholesterol in the liver tissue was extracted according to the kit (Puli), and the fat concentration in the tissue sample was corrected by the protein concentration of the sample.
  • the content of insulin (Millipore), alanine aminotransferase (ALT, Kehua), and arginine transaminase (AST, Kehua) in mouse serum was measured according to the kit.
  • mice The in vivo fat content, muscle to fat ratio (Lean/Fat) of the mice was determined using NMR instruments (The Minispec, Bruker) under conscious, non-invasive conditions.
  • DIO diet induced obesity, DIO mice were tested for pyruvate tolerance after taking Brazilian green propolis for 2 weeks. The results showed that Brazilian green propolis significantly inhibited hepatic gluconeogenesis in high fat-induced diabetic mice (DIO) (Fig. 1.D).
  • DIO mice After 3 weeks of taking the Brazilian green propolis, DIO mice found that propolis at the transcriptional level significantly inhibited the transcription levels of the hepatic gluconeogenesis rateers G6pc and Pck1 in the liver of DIO mice (Fig. 1.E).
  • Example 2 CREB/CRTC2 two-hybrid system for detecting the activity of Brazilian green propolis
  • Glucagon-induced hepatic gluconeogenesis is primarily dependent on the GCGR-cAMP-PKA-CREB/CRTC2-CRE signaling pathway (Fig. 2.A).
  • CRTC2 is activated by dephosphorylation of glucagon and binds to CREB through nuclear and CREB. It can strongly promote the transcription of genes related to hepatic gluconeogenesis. Based on this principle, CREB:CRTC2-Ser171Ala mammalian two-hybrid system was constructed. (Fig. 2.C) (This system uses the mutant CRTC2-Ser171Ala to mimic the dephosphorylated, activated CRTC2 state) and uses this system to examine the effect of propolis on CREB-CRTC2 binding.
  • propolis (0.2% (V/V) significantly inhibited the transcriptional activity of the reporter gene of the two-hybrid system (Fig. 2.C).
  • the results indicate that Brazilian propolis can inhibit hepatic gluconeogenesis at the transcriptional level by inhibiting the binding of CREB to CRTC2.
  • the inventors tracked the activity of the separated components of propolis.
  • P3 is the most active, and the activities of compounds P1, P2, P5, P6, P11 and P16 are relatively weak, while the compounds P4, P7, P10, P12, P14, P15, P17 and P19 are almost at 25 ⁇ M. There is no inhibition (Figure 2.G).
  • P1, P2, P3, P5 and P6 have similar structures (Fig. 2.H) and are all phenolic derivatives.
  • the results of evaluation of Brazilian green propolis monomer by CREB-CRTC2 two-hybrid system indicated that the active monomer of propolis was mainly concentrated in the methanol elution site of ethyl acetate layer, and P3 (Artepillin C) had the activity of inhibiting the binding of CREB/CRTC2. Propolis monomer compound.
  • CREB ⁇ Q1 full-length amino acids 102-341
  • CREB ⁇ Q1 ⁇ KID full length 160- 341 amino acids
  • CREB-bZIP full length 283-341 amino acids
  • Artepillin C inhibits the binding of CREB to CRTC2 by binding to the Q1 region of CREB.
  • the inhibitory activity of Artepillin C is dose-dependent and can exert inhibitory activity in hepatocytes.
  • Artepillin C significantly reduced glucagon-induced mRNA levels of hepatic gluconeogenesis genes G6pc, Pck1 and Pgc1a (Fig. 4.B).
  • CRTC2 acts as a transcriptional coactivator, which significantly promotes the transcriptional efficiency of CRE target genes. Therefore, the inventors used the deletion model to further detect whether Artepillin C inhibitory activity is dependent on the function of CRTC2. The results showed that the inhibitory activity of Artepillin C on hepatic glucose output disappeared in CRTC2 -/- primary hepatocytes (Fig. 4.G). At this time, Artepillin C also failed to inhibit glucagon-induced transcription of the hepatic gluconeogenesis key genes Pgc1 ⁇ and Pck1 (Fig. 4.H). This indicates that the inhibitory activity of Artepillin C on hepatic gluconeogenesis depends on the presence of CRTC2.
  • Artepillin C lowers blood lipid levels and lowers fat content in liver tissue, suggesting that Artepillin C may lower blood lipid levels mainly due to decreased hepatic lipid output, suggesting that Artepillin C inhibits hepatic lipid synthesis, output, and absorption. .
  • the present inventors used high-fat obese mice (DIO) and db/db mice as experimental subjects, and were administered by intraperitoneal injection (DIO: 0, 10, 20 mg/kg; db/db: 20 mg/kg).
  • the stability of the body inhibits the migration of SREBPs precursor proteins from the endoplasmic reticulum to the Golgi apparatus, thereby inhibiting the protein digestion process of SREBPs on the Golgi apparatus, thereby reducing the protein content of mature SREBPs.
  • Artepillin C inhibits the expression of the low-density protein cholesterol receptor (Ldlr) and inhibits the expression of the Ldlr receptor-degrading protein Pcsk9 by reducing the expression of Hmgcl and Hmgcs, the key rate-limiting enzymes in the cholesterol synthesis pathway (Fig. 6.C), and inhibiting the liver.
  • the synthesis and absorption of cholesterol reduces the synthesis and content of cholesterol in the liver; in the aspect of fatty acid metabolism, Artepillin C inhibits the synthesis of fatty acids.
  • Transcription of the key enzyme Fasn, Acc reduces fatty acid synthesis, inhibits fatty acid secretion by inhibiting ApoE transcription, inhibits excessive oxidation of fatty acids and synthesis of long-chain fatty acids by inhibiting Acox1 and Acsl1 expression (Fig. 6.C).
  • Fig. 7.A Three days after intraperitoneal injection of Artepillin C (20 mg/kg) in DIO mice, liver imaging analysis of DIO mice showed that Artepillin C inhibited starvation-induced G6P-LUC reporter transcriptional activity (Fig. 7.A). At the same time, Artepillin C significantly reduced the mRNA levels of hepatic gluconeogenesis genes Pgc1 ⁇ , Pck1 and G6pc in DIO liver tissue (Fig. 7.B). In the living liver tissues of DIO mice, the protein levels of the hepatic glucone-limiting enzyme PEPCK and the transcription factor PGC1 ⁇ were significantly inhibited by Artepillin C (Fig. 7.C).
  • the inventors further examined the effect of Artepillin C on overall glucose metabolism in mice.
  • healthy adult rats were given intraperitoneal injection of Artepillin C (20 mg/kg) three times, and elevated blood glucose levels stimulated by pancreatic hyperglycemia were significantly inhibited by Artepillin C (Fig. 7.D). Therefore, the effect of Artepillin C on glucose metabolism under insulin resistance was further examined.
  • the results showed that Artepillin C administered for one week significantly inhibited DIO Starvation blood glucose levels in mice (Fig. 7.E) and db/db mice (Fig. 7.F). After 5 weeks of administration, Artepillin C had no significant effect on serum insulin levels in db/db mice (Fig. 7.G).
  • insulin resistance is not only the pathogenesis and diagnosis of type 2 diabetes, but also the main link of many metabolic diseases, insulin resistance is the common pathophysiological basis of obesity, hyperglycemia, hyperlipidemia and metabolic syndrome.
  • the inventors further examined the effect of Artepillin on insulin sensitivity in diabetic mice. DIO mice and db/db mice were used as experimental subjects, and tolerance tests were performed 5 weeks after intraperitoneal injection (20 mg/kg). The results showed that Artepillin C significantly increased glucose tolerance (GTT) in diabetic mice (Fig.
  • Artepillin C significantly improved the balance of glycolipid metabolism in diabetic mice at the overall level, significantly increasing insulin sensitivity in diabetic mice.
  • the inventors' experimental results show that the small molecule compound Artepillin C (APC) inhibits the binding of CRTC2 and CREB after nuclear entry by directly binding to the N-terminus of CREB, and reduces the expression of downstream genes driven by CREB/CRTC2. It includes PGC1 ⁇ , the main component of hepatic gluconeogenesis, and important hepatic gluconeogenesis inhibitors, G6PC and PEPCK, which inhibit hepatic gluconeogenesis and liver glucose output and reduce starvation.
  • APC Artepillin C
  • APC inhibits the expression of SREBPs driven by LXRE, inhibits the expression of SREBPs driven by LXRE, inhibits the downstream target genes of SREBPs, and lowers the cholesterol regulated by SREBPs by inhibiting the expression of nuclear receptor LXR ⁇ and inhibiting the transcriptional coactivator PGC1 ⁇ . Fatty acid synthesis, absorption, and ultimately reduce blood fat content (Figure 9).
  • APC significantly improves hyperglycemia and hyperlipidemia in diabetic mice and reduces fat, APC regulates glucose homeostasis as a whole, relieves insulin resistance and enhances overall insulin sensitivity. .
  • the compound 63#, 65# also had an activity of inhibiting the binding of CREB/CRTC2 to each other, as shown in Fig. 10.
  • 65# has strong inhibitory activity, and the IC50 of the 65# compound is about 15.9 uM by the two-hybrid test.

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Abstract

La présente invention concerne une utilisation de l'artépilline C et d'analogues de celle-ci pour la prévention et le traitement de maladies métaboliques. La présente invention est la première à décrire un effet significatif d'artépilline C ou d'analogues de ceux-ci dans la prévention et le traitement de maladies métaboliques, de manière à obtenir un médicament multifonctionnel focalisé sur l'ajustement de CREB/CRTC2 ou SREBP.
PCT/CN2016/081956 2015-06-04 2016-05-13 Utilisation d'artépilline c et d'analogues de ceux-ci dans la préparation de médicaments pour la prévention et le traitement de maladies métaboliques Ceased WO2016192523A1 (fr)

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CN110917184A (zh) * 2019-12-16 2020-03-27 中国科学院昆明植物研究所 香豆酸类化合物的应用、药物组合物及其应用
CN112280853B (zh) * 2020-11-18 2022-07-22 武汉大学 一种肥胖表型分子标志物及其应用

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