WO2021218249A1 - Dérivé de spinosyne en tant qu'activateur de l'argininosuccinate synthétase et son application - Google Patents

Dérivé de spinosyne en tant qu'activateur de l'argininosuccinate synthétase et son application Download PDF

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WO2021218249A1
WO2021218249A1 PCT/CN2021/073695 CN2021073695W WO2021218249A1 WO 2021218249 A1 WO2021218249 A1 WO 2021218249A1 CN 2021073695 W CN2021073695 W CN 2021073695W WO 2021218249 A1 WO2021218249 A1 WO 2021218249A1
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ass1
plasmid
spinosyn
protein
carbon
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罗志勇
刘苏友
邹自征
胡息源
罗眺
陈筱丹
孔繁蓉
罗均利
罗文松
马大友
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中南大学
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/04—Other carbon-nitrogen ligases (6.3.4)
    • C12Y603/04005—Argininosuccinate synthase (6.3.4.5)

Definitions

  • the present invention relates to spinosyn A (Spinosyn A) and its derivatives which can regulate and activate Argininosuccinate synthetase (Argininosuccinate synthesis 1, ASS1).
  • spinosyn A Spinosyn A
  • Argininosuccinate synthesis 1, ASS1 Argininosuccinate synthesis 1, ASS1
  • Such compounds can be used as treatments for diseases related to succinate synthase deficiency, such as anti-tumor and citrullinemia, and belong to the field of medicine.
  • Argininosuccinate synthase (Argininosuccinate synthase, ASS1; EC 6.3.4.5) was first found in the liver, and was later considered to be a ubiquitous enzyme in mammals.
  • the ASS1 gene is located on chromosome 9q34.11, the gene length is 56kb, the open reading frame length is 1239bp, with 16 exons, it encodes 412 amino acids, and the molecular weight is 46kDa.
  • ASS1 catalyzes citrulline and aspartic acid to produce arginine succinate under the condition of ATP function. The compound is further decomposed into arginine and fumaric acid under the action of arginine succinate lyase.
  • arginine Amino acid further enters the urea cycle or is used in metabolic processes such as protein synthesis.
  • the urea cycle can convert toxic ammonia into non-toxic urea to be excreted from the body, which is the main way to detoxify ammonia in the body.
  • ASS1 expression is down-regulated or mutated, its enzyme catalytic activity decreases or lacks, which will block the urea cycle and increase the level of citrulline.
  • Arginine succinate synthase is a key enzyme of the urea cycle (see Figure 2).
  • aspartic acid is one of the key substrates of ASS1, and the level of ASS1 expression and its activity will determine its use in the urine cycle. The utilization of aspartic acid. Low expression of ASS1 will limit the utilization of aspartic acid.
  • Citrullinemia (Citrullinemia, CTLN) is an autosomal recessive inherited disorder of the urea cycle. The main clinical manifestations are increased citrullinemia and hyperammonemia. According to the different pathogenesis, it is divided into type I and II type. Citrullinemia type I (Citrullinemia type 1, CTLN 1) is caused by ASS1 gene defect, with an incidence rate of about 1/250,000, which is the third largest urea cycle disorder; type II citrullinemia (Citrullinemia type 1) 2. CTLN 2) It is caused by the mutation of Citrin gene. CTLN1 is mainly a toxic phenomenon of hyperammonemia in clinical manifestations.
  • citrullinemia occurs in the neonatal period, which is characterized by hyperammonemia accompanied by neurological function decline, poor prognosis, and high mortality; delayed-onset citrullinemia has a later onset and mild symptoms. May exhibit recurring neurological symptoms, such as drowsiness, mental retardation, etc. Some patients have no symptoms and only the biochemical phenotype detected during newborn screening.
  • mutations that cause defects in the arginine succinate synthase gene of CTLN1 There are many mutations that cause defects in the arginine succinate synthase gene of CTLN1. There are 137 mutations that have been reported, mainly missense mutations. In addition, a few cases have nonsense mutations, abnormal splicing and deletion mutations. The first eight most frequently occurring mutation types are p.Gly390Arg, p.Trp179Arg, p.Gly362Val, p.Arg363Trp, p.Gly324Ser, p.Arg157His, p.Arg304Trp, and p.Val263Met, corresponding to 124, 27, and p.Val263Met. 24, 17, 16, 14, 13, and 12 cases.
  • Citrullinemia is a disease of chromosomal abnormalities. There is currently no cure. The treatment is mainly low-protein diet and blood ammonia-lowering treatment. If the symptoms are more serious or the blood ammonia is too high, it depends on blood or Abdominal dialysis treatment. Therefore, the invention of drugs for the treatment of citrullinemia has very important clinical value.
  • CAD Carbamoylphosphate synthetase II (CPSase), Aspartate transcarbamoylase (ATCase) and Dihydroorotate hydrogenase (DHOase)] are pyrimidines
  • CPSase Carbamoylphosphate synthetase II
  • ATCase Aspartate transcarbamoylase
  • DHOase Dihydroorotate hydrogenase
  • the key rate-limiting enzyme for de novo nucleotide synthesis is as follows: glutamine and carbon dioxide are powered by ATP in the cytosol, and carbamoyl phosphate synthase II is catalyzed to generate carbamoyl phosphate.
  • aspartate transcarbamylase catalyzed by aspartate transcarbamylase to transfer the carbamoyl group to the amino group of aspartic acid to generate carbamoyl aspartic acid.
  • Carbamyl aspartic acid is dehydrated and cyclized to produce dihydroorotic acid, which is then dehydrogenated to form orotic acid.
  • Orotic acid is an essential precursor of pyrimidine nucleotides. It can be seen that aspartic acid is also a key substrate of CAD. If tumor cells proliferate abnormally, more aspartic acid must be involved in the synthesis of nucleotides.
  • Aspartic acid contains two carboxyl groups and is highly polar. It is difficult for exogenous aspartic acid such as food to enter cells. The source of intracellular aspartic acid depends on its endogenous biosynthesis. The catabolic pathway of aspartic acid determines its role and function in cells. Aspartic acid is the common substrate of ASS1 and CAD, therefore, both ASS1 and CAD use aspartic acid competitively.
  • ASS1 is closely related to tumor growth. In some tumors, ASS1 expression is down-regulated or defective, including breast cancer, melanoma, hepatocellular carcinoma, prostate cancer, bladder cancer, mesothelioma, ovarian cancer, kidney cancer, and pancreatic malignancies , Nasopharyngeal carcinoma, osteosarcoma and myxofibrosarcoma. There is an obvious correlation between ASS1 deficiency and poor prognosis of cancer.
  • ASS1 has a tumor suppressor function in a variety of tumors.
  • activating ASS1 can inhibit aspartic acid Pathway to synthesize pyrimidine nucleotides necessary for tumor cell proliferation. Therefore, we believe that ASS1 protein can become a potential direct target of anti-tumor drugs. So far, there is no public report in the literature about small chemical molecules that can modulate the activity of ASS1.
  • Spinosyn is an intracellular secondary metabolite produced by Saccharoplyspora spinosa through aerobic fermentation. It is a macrolide antibiotic with insecticidal activity. Spinosyn is traded under the name Spinosad (SP), and its main active ingredients are A (Spinosyn A, SPA, 85-90%) and D (Spinosyn D, 10-15%). Spinosyn contains a unique four-ring structure that connects Two different six-membered sugars. As a broad-spectrum biological pesticide, SP mainly controls Lepidoptera and Thysanoptera pests.
  • the present invention discloses spinosyn derivatives as ASS1 activators, which can activate ASS1 and mutant ASS1 G362V enzyme activities, and are used in drugs for treating diseases related to ASS1 deficiency, especially citrullinemia and anti-tumor.
  • the technical problem solved by the present invention is to provide a new use of a class of compounds, which belong to spinosyn derivatives and can be used as arginine succinate synthase (ASS1) activators.
  • the technical solution of the present invention is to provide a spinosyn derivative and a medically acceptable salt thereof as an activator of arginine succinate synthase (ASS1).
  • the spinosyn derivative has a structure General formula (I):
  • R1 is selected from the following II-VIII groups:
  • R8 and R9 are all independently selected from hydrogen, 1-20 carbon atoms (preferably 2-16 carbon atoms, more preferably 2-10 carbon atoms), 1-20 carbon atoms Haloalkyl (preferably 2-16 carbon haloalkyl, more preferably 2-10 carbon haloalkyl), 1-10 carbon atom alkyl substituted by 1-6 carbon alkylamino group (preferably 2-6 Alkyl with carbon atoms), hydroxyalkyl with 1-10 carbon atoms substituted by acyloxy (preferably hydroxyalkyl with 2-6 carbon atoms), arylmethyl, phosphoryl, 1-10 carbon atoms Alkanoyl (preferably alkanoyl with 2-6 carbon atoms), aroyl, Wherein, J is selected from halogen atoms, R19R20N-, tetrahydropyrrolyl, piperidinyl, morpholinyl, piperazinyl, Wherein R16 is selected from hydrogen, an alkyl group of 1-10 carbon atoms (preferably an
  • R10, R11, and R12 are all independently selected from hydrogen, 1-20 carbon alkyl group (preferably 2-16 carbon atom alkyl group), 1-20 carbon alkenyl group (preferably 2-16 carbon atom Alkenyl, more preferably alkenyl with 2-10 carbon atoms), arylmethyl;
  • R13 is selected from hydrogen, R14R15N-, nitrogen-containing heterocycle, oxygen-containing heterocycle, sulfur-containing heterocycle, and phosphorus-containing heterocycle;
  • R14, R15, R19, and R20 are all independently selected from hydrogen, an alkyl group of 1 to 6 carbon atoms, and an alkyl group of 1 to 10 carbon atoms substituted by an amino group;
  • R2 is selected from ethyl, propyl, butyl, 3-4 carbon alkenyl
  • R3 is selected from hydrogen and methyl
  • R5, R6, and R7 are all independently selected from hydrogen, 1-3 carbon alkyl, acetyl, propionyl;
  • W is selected from CH 2 , O, NH, S;
  • X is an anion
  • X is an anion, chlorine, bromine, iodine, sulfate, hydrogen sulfate, phosphate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, hydroxide;
  • n is an integer of 0-4, and m is an integer of 0-20.
  • R2 is ethyl
  • R4 is hydrogen
  • R5, R6, and R7 are all independently selected from methyl or ethyl.
  • the W is selected from O, NH, NCH 3 , and S.
  • nitrogen-containing heterocycle, oxygen-containing heterocycle, sulfur-containing heterocycle, and phosphorus-containing heterocycle respectively mean that the heteroatoms in the heterocycle are nitrogen, oxygen, sulfur, and phosphorus.
  • heteroatoms in the nitrogen-containing heterocycle are nitrogen atoms, and the number is 1-3.
  • nitrogen-containing heterocyclic ring is tetrahydropyrrolyl, piperidinyl, morpholinyl, piperazinyl, Wherein R16 is selected from alkyl groups of 1-10 carbons.
  • the spinosad derivative has the structure:
  • the present invention relates to the compound of general formula (I), which is characterized by spinosyns and derivatives thereof involved in patents CN201610355188.0, ZL201010123056.8, CN201610356840.0 and US6001981.
  • the present invention also provides the application of the small molecule composition comprising the general formula (I) as an arginine succinate synthetase.
  • the compound may also contain one or more pharmaceutically acceptable carriers or excipients
  • the pharmaceutical composition of the present invention contains spinosyn and its derivatives represented by the general structural formula (I), and may also contain one or more pharmaceutically acceptable carriers or excipients.
  • the carrier or excipient may include glycerol, ethanol, buffered saline, physiological saline, and combinations thereof.
  • the pharmaceutical composition may also include penetration enhancers, antioxidants, and the like.
  • the present invention provides the use of spinosyn and its derivative LM-2I as represented by the general formula (I) in the prevention and treatment of diseases associated with low ASS1 expression or mutant ASS1G362V.
  • ASS1 low expression or mutant ASS1G362V related diseases include but are not limited to type I citrullinemia and related cancers.
  • the present invention uses the biotin probe method to identify small molecule targets, and finds that the target of the compound of the general structural formula (I) is ASS1.
  • the inventors synthesized and discovered spinosyn A-biotin probes, such as Xn-03-17A (abbreviated as 17A), and found that it has good anti-tumor proliferation activity and can replace drugs for target experiments.
  • Xn-03-17A for target identification, and the results showed that the target of spinosyn derivatives was the protein ASS1.
  • the ASS1 monoclonal antibody was used for Western blot analysis and combined with mass spectrometry to confirm that it was ASS1.
  • the present invention determines the ASS1 activation activity of spinosyn derivatives according to the principle and method of arginine succinate synthetase (ASS1) activity measurement.
  • the principle of ASS1 activity detection is: ASS1 catalyzes the reaction of citrulline and aspartic acid to produce arginine succinic acid. This reaction consumes ATP to generate pyrophosphate (PPi), and the generated PPi is produced under the catalysis of pyrophosphatase. Phosphoric acid, phosphoric acid reacts with ammonium molybdate to generate phosphomolybdenum heteropoly acid, which is reduced by vitamin C to generate blue phosphomolybdenum blue, which has a characteristic absorption peak at OD660.
  • the results showed that spinosyn derivatives can increase the enzyme activity of ASS1 to varying degrees. Compared with the positive control of ASS1, the enzyme activity increased by 5.85-203.86%.
  • the MTT experiment is used to screen the activity of spinosyn derivatives on a variety of human tumor cells.
  • the low expression of ASS1 is related to the low overall survival rate and low disease-free survival rate of breast cancer patients.
  • ASS1 is an independent prognostic factor of breast cancer overall survival rate and disease-free survival rate, and can be used as a new candidate molecular marker for breast cancer.
  • the present invention found that spinosyn and its derivative LM-2I can increase the ASS1 G362V enzyme activity to varying degrees through the ASS1 in vitro enzyme activity experiment.
  • spinosyn and its derivatives are the first discovered mutant arginine succinate targeting the tumor suppressor protein arginine succinate synthase 1 (ASS1) and type I citrullinemia A type of activator of acid synthase ASS1 G362V . Because ASS1 is generally down-regulated or defective in tumors, spinosyn and its derivatives have significant anti-tumor effects. While for congenital genetic diseases due -I citrullinemia type ASS1 ASS1 G362V mutation resulting difficult to cure, spinosyn and derivatives thereof can also be used as orphan drugs by reversing and restoring functional activity ASS1 G362V play a significant effect . Spinosyn and its derivatives are mainly used as drugs for the treatment of diseases related to ASS1 deficiency, especially for type I citrullinemia and anti-tumor therapy.
  • Figure 1 shows the general structural formula of spinosyn derivatives
  • FIG. 2 shows the catalytic reaction process of arginine succinate synthase 1 (ASS1)
  • Figure 3 shows the pathway of aspartic acid metabolism
  • Figure 4 shows the identification of the target of the compound; in the figure, 1A) Silver staining result of Pull-down product in cell lysate; 1B) Western blot detection of ASS1 antibody;
  • Figure 5 shows the mass spectrometry identification probe Biotin-SPA bound protein as ASS1;
  • Figure 6 shows the relative catalytic activity of different compounds on ASS1
  • Figure 7 shows Coomassie brilliant blue staining after purification of ASS1 WT and ASS1 G362V proteins
  • Figure 8 shows the effects of spinosyn A (SPA) and LM-2I on the activity of mutant ASS1;
  • Figure 9 shows that there are differences in the expression of ASS1 in multiple breast cancer cell lines.
  • Figure 10 shows that the proliferation rate of breast cancer multi-cell lines is significantly negatively correlated with the expression of ASS1.
  • MTT method was used to detect the 48h proliferation rate of multi-cell lines (a), (b) the correlation analysis between cell proliferation rate and ASS1 expression;
  • Figure 11 shows the detection of drug sensitivity after knockdown or overexpression of ASS1.
  • MDA-MB-231ASS1 overexpression cell line (a) or MCF-7 ASS1 knockdown cell line (b) were treated with SPA or LM-2I for 48h, and cell survival rate was detected by MTT.
  • Figure 12 shows that SPA and LM-2I inhibit tumor volume growth.
  • SPA (10mg/kg/d) and LM-2I (5m/kg/d) were administered for 18 days, every other day, and the longest diameter and shortest diameter of the tumor were recorded, and the tumor volume was calculated by the formula, where Vehicle is a solvent control.
  • Knockout of ASS1 can significantly reduce the anti-tumor activity of SPA and LM-2I.
  • CRISPR/Cas9 was used to knock out ASS1(a), SPA(b) and LM-2I(c) in MDA-MB-231 cells for 48h, and the survival rate was detected by crystal violet staining. Each experiment was repeated three times.
  • SPA and LM-2I inhibit the growth of tumor volume.
  • SPA (10mg/kg/d) and LM-2I (5m/kg/d) were administered for 28 days, every other day, and the longest diameter and shortest diameter of the tumor were recorded, and the tumor volume was calculated by the formula, where Vehicle is a solvent control.
  • Vehicle is a solvent control.
  • the number of mice in each sample group is 8 and the statistics are performed by one-way analysis of variance. * means P ⁇ 0.05, *** means P ⁇ 0.001.
  • FIG. 4A is the result of silver staining of Pull-down product from MCF-7 cell lysate using Xn-03-17A probe.
  • Figure 4B is a Western blot detection image using ASS1 antibody. This issue clearly proves that ASS1 is the target of spinosyn derivatives.
  • ASS1 catalyzes the reaction of citrulline and aspartic acid to produce arginine succinic acid. This reaction consumes ATP to generate pyrophosphate (PPi), and the generated PPi is produced under the catalysis of pyrophosphatase.
  • Phosphoric acid, phosphoric acid reacts with ammonium molybdate to generate phosphomolybdenum heteropoly acid, which is reduced by vitamin C to generate blue phosphomolybdenum blue, which has a characteristic absorption peak at OD660.
  • the enzyme activity is: [(drug+ASS1)OD 660 -NCOD 660 ]/(ASS1OD 660 -NCOD 660 ) ⁇ 100%.
  • (Drug + ASS1) OD 660 the absorbance of the solution when the drug and ASS1 are added (wavelength 660nm); ASS1OD 660 : the absorbance of the solution when ASS1 is added, the absorbance of the solution (wavelength 660nm); NCOD 660 : the absorbance of the blank reference solution (wavelength 660nm).
  • the total RNA of human breast cancer cell line MCF-7 cell line was extracted, and the ASS1 gene was amplified by RT-PCR, using primers (5′-3′):
  • ASS1-F ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC (SEQ ID NO10);
  • ASS1-R AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT
  • the pET-28a plasmid was extracted, digested with restriction enzymes, and recovered by the gel. According to the method of homologous recombination, the ASS1 gene was integrated into the pET-28a plasmid.
  • Transformation Transform the constructed pET-28a plasmid into Escherichia coli DH5a, select positive clones and send them for sequencing. After the clones with correct sequencing are expanded and cultured, the plasmids are extracted and transformed into the BL21(DE3) strain. The positive clones are identified by PCR and confirmed The band size is between 1.0-1.5kb and is sent for sequencing. Select the correctly sequenced strains to preserve the bacteria at -80°C and use them for later use.
  • Prokaryotic expression Take out the constructed ASS1BL21(DE3) strain stored at -80°C and restore it to OD660 to 0.4-0.6, add IPTG to a final concentration of 1mM and induce 4h, collect the bacterial solution by centrifugation, and store at -80°C spare.
  • Protein extraction Thaw the bacteria liquid stored at -80°C on ice, add 2-5mL per gram of lysis buffer to resuspend the bacteria, add lysozyme to a final concentration of 1mg/mL, place on ice for 30 minutes, and ultrasonically break the wall , Centrifuge, take the supernatant, set aside on ice.
  • the buffer components are: 20mM Tris . HCl (pH 7.8), 2mM ATP, 10mM citrulline, 10mM aspartic acid, 6mM MgCl2, 20mM KCl, 0.2U pyrophosphatase.
  • Molybdate buffer components are: 10mM vitamin C, 2.5mM ammonium molybdate, 2% (V/V) sulfuric acid (enzyme activation results are shown in Figure 6 and Table 1).
  • ASS1 pET28a plasmid In order to detect the effect of SPA and its derivative LM-2I on the enzyme activity of protein ASS1 and ASS1 G362V , we constructed the ASS1 pET28a plasmid through homologous recombination technology, and on this basis, used gene-directed mutagenesis technology to construct the ASS1 G362V pET28a plasmid.
  • the constructed plasmid was transformed into BL21 (DE3) for prokaryotic expression and purified protein ASS1 and ASS1 G362V .
  • amino acid sequence of ASS1 is:
  • ASS1 is G at position 362, and ASS1G 362V is V in brackets at position 362.
  • ASS1-F ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC (SEQ ID NO 3);
  • ASS1-R AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT (SEQ ID NO 4).
  • Transformation Transform the constructed pET-28a plasmid into Escherichia coli DH 5 a, and select positive clones for sequencing. After the clones with correct sequencing are expanded and cultured, the plasmids are extracted and transformed into BL21(DE 3 ) strain. The positive clones were identified by PCR, and the band size was determined to be between 1.0-1.5 kb and sent for sequencing.
  • the nucleotide sequence of ASS1 is exactly the same as the CDS sequence of ASS1variant 1 (ACCESSION: NM_000050) in the NCBI database.
  • its sequence is exactly the same as the ASS1 sequence in the Uniprot database. Therefore, the prokaryotic expression of ASS1 we constructed
  • the carrier can be used for subsequent research. Select the correctly sequenced strains to preserve the bacteria at -80°C and use them for later use.
  • Design point mutation primers, and design mutation primers based on the principle of homologous recombination are designed point mutation primers, and design mutation primers based on the principle of homologous recombination:
  • ASS1 G362V -F TACATCCTCGTCCGGGAGTCCCCACTGTCTCTCTACAAT (SEQ ID NO 5)
  • ASS1 G362V- R GGACTCCCGGACGAGGATGTACACCTGGCCCTTGAGGAC (SEQ ID NO 6)
  • PCR amplification use mutant primers to amplify the pet28a-ASS1 plasmid; digest the amplified product with Dpnl to remove the methylated template plasmid; perform homologous recombination, transformation, and sequencing for identification.
  • Prokaryotic expression Take out the constructed ASS1BL21(DE 3 ) strain stored at -80°C and restore it to OD 660 to 0.4-0.6, add IPTG to a final concentration of 1 mM for induction for 4 hours, and collect the bacterial solution by centrifugation, -80 Store at °C for later use.
  • Lysis buffer (1L) add protease inhibitor before use, add 5mM mercaptoethanol before use: 50mM Tris.HCl, 500mM NaCl, 10mM imidazole, Adjust pH to 8.0 using NaOH;
  • Wash buffer (1L) (without protease inhibitor, add 5mM mercaptoethanol before use): 50mM Tris.HCl, 500mM NaCl, 20mM imidazole, Adjust pH to 8.0 using NaOH;
  • Elution buffer (1L) (no protease inhibitor, no 5mM mercaptoethanol): 50mM Tris.HCl, 500mM NaCl, 250mM imidazole, Adjust pH to 8.0 using NaOH
  • the concentration of ASS1 and ASS1 G362V is 0.5 ⁇ M, and the concentration of spinosyn and its derivative LM-2I is 10 ⁇ M. Incubate overnight at 4°C, add buffer, incubate at 37°C for 1 min, and then add an equal volume of Molybdate buffer to develop color for 3 min. At 37°C, the absorbance was detected by OD 660.
  • the ASS1 group without drug incubation was used as a positive control, and the reagent group without ASS1 was NC.
  • the relative enzyme activity is: [ASS1 G362V OD 660 -NCOD 660 ]/(ASS1OD 660 -NCOD 660 ) ⁇ 100%.
  • the buffer components are: 20mM Tris.Hcl (pH 7.8), 2mM ATP, 10mM citrulline, 10mM aspartic acid, 6mM MgCl2, 20mM KCl, 0.2U pyrophosphatase.
  • Molybdate buffer components are: 10mM vitamin C, 2.5mM ammonium molybdate, 2% (V/V) sulfuric acid.
  • ASS1 Through the in vitro enzyme activity experiment of ASS1, it was found that SPA and LM-2I can restore the activity of mutant ASS1.
  • the reaction concentration of ASS1 and ASS1 G362V was 0.5 ⁇ M
  • the concentration of Spinosyn A (SPA) and its derivatives LM-2I was 10 ⁇ M
  • the reaction time was 1 min.
  • ASS1 alone was a positive control.
  • the overexpression vector and shRNA interference vector of ASS1 were purchased from Gemma Gene. There are two shRNAs, the sequences are (5′-3′):
  • ASS1 shRNA1 ASS1 shRNA1:
  • the ASS1 overexpression sequence is obtained by a fully synthetic method, and its sequence is the CDS sequence of human ASS1 variant 1 (ACCESSION: NM_000050).
  • 293T tool cells were cultured to 90% confluence, trypsinized, resuspended after centrifugation, and counted.
  • MDA-MB-231 and MCF-7 cells Resuscitate MDA-MB-231 and MCF-7 cells, culture them in a 10cm petri dish until they are in good condition. When the cells grow to be 90% confluent, digest, centrifuge and count, MDA-MB-231 (1 ⁇ 10 6 /dish) Passage with MCF-7 (1.5 ⁇ 10 6 /dish), and when the cell density reaches 40-50%, perform the lentivirus infection experiment.
  • the surviving cells are digested, counted, and 0.5 cells per well are seeded into a 96-well plate for monoclonal screening.
  • the grown monoclonal trypsin is digested and then inoculated into a 6-well plate. When it grows to about 80-90%, half is used for passaging, and half is used for protein extraction and Western blot to detect the expression of the target protein.
  • the single clones that meet the requirements are amplified and cultured, stored in liquid nitrogen, and MTT experiments are performed.
  • MTT method cell experiment use ASS1 knockdown or overexpression cell line to detect the drug sensitivity of spinosyn A (SPA) or LM-2I, and the same concentration of SPA or LM-2I can simultaneously treat MCF-7 ASS1 sh and MCF-7 NC or MDA-MB-231 ASS1 OE and MDA-MB-231 NC cells for 48h.
  • SPA spinosyn A
  • Tumors with low ASS1 expression are not limited to breast cancer, such as melanoma, hepatocellular carcinoma, prostate cancer, bladder cancer, mesothelioma, ovarian cancer, kidney cancer, pancreatic malignancies, nasopharyngeal carcinoma, osteosarcoma and myxofibrosarcoma.
  • the triple-negative breast cancer MDA-MB-231 cell line with low ASS1 expression, high malignancy, easy metastasis, poor prognosis, and lack of targeted drug therapy was selected as a representative to construct a nude mouse xenograft model for in vivo experiments.
  • each nude mouse was injected with 5 ⁇ 10 6 MDA-MB-231 cells into the armpit of the mouse. After 4-7 days, it will grow into a rice grain-sized tumor.
  • the tumor is about 100mm 3 , which means that the modeling is successful.
  • the solvent group is a negative control.
  • the concentration of the SPA treatment group is 10 mg/kg/d, and the concentration of the LM-2I treatment group is 5 mg/kg.d.
  • the tumor volume of Vehicle group was 841.52 ⁇ 420.81mm 3 ; the volume of SPA (10mg/kg/d) was 386.27 ⁇ 77.06mm 3 , which was 54.10% lower than that of Vehicle, which was statistically different; LM-2I (5mg/kg /d) The volume of the group was 306.41 ⁇ 79.08mm 3 , which was reduced by 63.60% compared with Vehicle, which was statistically different. From the point of view of the inhibitory effect, LM-2I (5mg/kg/d) is better than SPA (10mg/kg/d), and the drug concentration of LM-2I is only half of that of SPA ( Figure 12).
  • ASS1-sgRNA1 ASS1-sgRNA1:
  • ASS1-sgRNA2 ASS1-sgRNA2:
  • R AAACGGACACCTCGTGCATCCTCGC (SEQ ID NO 15);
  • the designed paired primers are annealed to form a double-strand for use.
  • the gene knockout plasmid lentiCRISPR v2 is digested with BsmBI-v2, electrophoresis, and the gel is recovered and ligated with the paired primers.
  • the constructed plasmid is transferred to Stab13, and the sequence is correct for use;
  • the experimental group using ASS1-sgRNA1 primer to knock out ASS1 is ASS1-KO1 MDA-MB-231; the experimental group using ASS1-sgRNA2 primer to knock out ASS1 is ASS1-KO2 MDA-MB-231; the blank experimental group without ASS1 knocking out It is ASS1-NC (Negative Control) MDA-MB-231 cells. Two pairs of different primers were used to construct knockout ASS1 vectors, which fully verified the accuracy of the test.
  • Virus packaging refer to Example 4 for specific test steps
  • MTT detection refer to Example 4 for specific test steps
  • mice were purchased from SJA Laboratory Animal Co., Ltd (Hunan), and they were all 4-6 week old female mice, weighing 18-22 g.
  • mice were raised in an SPF environment at 23-24°C, and fed with sterilized feed and water. After feeding for 3-4 days, the mice in good condition were marked.
  • MDA-MB-231 cells were cultured to logarithmic growth phase, 0.25% trypsinization, 800rpm centrifugation, room temperature for 10 minutes, after collecting the cells, wash the cells with serum-free DMEM/F12 (1:1) medium for 3 times After centrifugation at 800 rpm, the cells were collected for 10 minutes at room temperature, and then a small amount of serum-free DMEM/F12 (1:1) medium was added to resuspend the cells. After counting, the cell density was adjusted to 5 ⁇ 10 7 cells/mL.
  • the needle inlet should be kept at a distance of about 1 cm from the site of tumor formation. In about 5-7 days, the tumor volume reaches about 100mm 3 . Observe whether there is a stable nodule formation. If there is, it means that the model is successfully constructed, and subsequent experiments can be carried out.
  • mice with little difference in body weight and tumor size were randomly divided into Vehicle group, SPA group and LM-2I group, each with 8 mice.
  • SPA 10mg/kg/d
  • LM-2I 5mg/kg/d
  • subcutaneous injection taking into account the poor water solubility of SPA, the solvent we used It is DMSO: Tween 80 (1:1)
  • each administration dose is 0.1mL, administered every other day, and measure the weight of the mouse and the length of the tumor, observe the life of the mouse and record whether there is any abnormality. Click the formula to calculate the tumor
  • Body volume (a is the longest diameter of the tumor, b is the shortest diameter).
  • the tumor volume of Vehicle group was 1076.65 ⁇ 230.41mm 3 ; the volume of SPA (10mg/kg/d) was 259.48 ⁇ 88.82mm 3 , which was reduced by 75.90% compared with Vehicle, with statistical difference; LM-2I( The volume of 5mg/kg/d) group was 143.91 ⁇ 70.42mm 3 , which was reduced by 86.63% compared with Vehicle, which was statistically different.
  • LM-2I (5mg/kg/d) is better than SPA (10mg/kg/d), and the drug concentration of LM-2I is only half of that of SPA ( Figure 14).

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Abstract

La présente invention concerne la spinosyne A et un dérivé de celle-ci en tant qu'activateurs de l'argininosuccinate synthétase 1 (ASS1) et de l'ASS1 mutante G362V et une application de celle-ci, le dérivé de spinosyne ayant la formule de structurale générale (I). La spinosyne et son dérivé peuvent être utilisés en tant que médicament pour le traitement de la citrullinémie de type I et en tant que médicament antitumoral par ciblage de l'activation d'ASS1 et d'ASS1 mutante G362V.
PCT/CN2021/073695 2020-04-30 2021-01-26 Dérivé de spinosyne en tant qu'activateur de l'argininosuccinate synthétase et son application WO2021218249A1 (fr)

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EP4079744A1 (fr) * 2021-04-23 2022-10-26 Acies Bio d.o.o. Nouveaux promédicaments de spinosyne
AR125402A1 (es) * 2021-04-23 2023-07-12 Acies Bio D O O Profármacos nuevos de espinosinas
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