CN111632061A

CN111632061A - Spinosad derivative serving as argininosuccinate synthetase activator and application thereof

Info

Publication number: CN111632061A
Application number: CN202010375736.2A
Authority: CN
Inventors: 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
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2020-04-30
Filing date: 2020-05-07
Publication date: 2020-09-08
Anticipated expiration: 2040-05-07
Also published as: CN111632061B; WO2021218249A1; CN114796254A

Abstract

The invention discloses spinosyn A and derivatives thereof as argininosuccinate synthetase 1(ASS1) and mutant argininosuccinate synthetase ASS1^G362VThe spinosyn derivative has a structural general formula (I):

the spinosad and the derivatives thereof are stimulated by targetingLive argininosuccinate synthetase 1(ASS1) and mutant argininosuccinate synthetase ASS1^G362VThereby being used as a medicine for treating I type citrullinemia and an anti-tumor medicine.

Description

Spinosad derivative serving as argininosuccinate synthetase activator and application thereof

Technical Field

The present invention relates to spinosyn A and derivatives thereof that modulate activation of Argininosuccinate synthase (ASS 1). The compounds can be used for treating diseases related to the defect of the amino acid-substituted succinic acid synthetase, such as tumor resistance and citrullinemia, and belong to the field of medicines.

Background

Argininosuccinate synthetase (Arginosinosccinate synthase, ASS 1; EC 6.3.4.5) was first found in the liver and later was considered to be a ubiquitous enzyme in mammals. The ASS1 gene is located on chromosome 9q34.11, has a gene length of 56kb, an open reading frame length of 1239bp, 16 exons, 412 coded amino acids and a molecular weight of 46 kDa. ASS1 catalyzes citrulline and aspartate to generate argininosuccinate under ATP functional conditions, and the compound is further decomposed into arginine and fumarate under the action of argininosuccinate lyase, wherein arginine further enters the urea cycle or is used for metabolic processes such as protein synthesis. The urea circulation can convert the toxic ammonia into non-toxic urea to be discharged out of the body, and is a main way for in vivo ammonia toxicity removal. If ASS1 is down-regulated or mutated, its enzymatic activity is reduced or absent, which will block the urea cycle and increase citrulline levels. Argininosuccinate synthetase (key enzyme in urea cycle) (see fig. 2) and aspartic acid as one of the key substrates of ASS1, the level of expression and activity of ASS1 will determine the availability of aspartic acid for use in the urinary cycle.

Citrullinemia (CTLN) is an autosomal recessive inherited urea cycle disorder, which is clinically manifested mainly by citrulline increase and hyperammonemia, and is classified into type i and type ii according to the difference in pathogenesis. Citrullinemia type i (ctrllinemia type 1, CTLN 1), which is caused by a defect in ASS1 gene, has an incidence of about 1/250,000, and is the third major urea cycle disorder; type II Citrullinemia (Citrulinemia type 2, CTLN 2) is caused by mutations in the Citrin gene. CTLN1 is clinically characterized mainly by hyperammonemia toxicity, and is classified into classical and delayed by the onset time and the severity of the disease. Typical citrullinemia occurs in the neonatal period and is characterized by hyperammonemia accompanied with the functional decline of a nervous system, poor prognosis and high mortality; late onset citrullinemia is associated with late onset and mild symptoms, patients may show recurrent neurological symptoms such as lethargy, intellectual disability, etc., and some patients have no symptoms and only have biochemical phenotypes detected in neonatal screening.

There are many mutations causing deficiency of the argininosuccinate synthetase gene of CTLN1, and there are 137 kinds of mutations reported so far, mainly missense mutations, and in addition, nonsense mutations, aberrant splicing and deletion mutations occur in a few cases. The first eight most frequently occurring mutation types were p.gly390arg, p.trp179arg, p.gly362val, p.arg363trp, p.gly324ser, p.arg157his, p.arg304trp, and p.val263met, corresponding to 124, 27, 24, 17, 16, 14, 13, and 12 cases, respectively. In the first eight-bit mutation with the highest mutation rate, the mutations p.Gly390Arg, p.Arg157His and p.Gly324Ser are completely inactivated in vitro experiments, while the mutations p.Trp179Arg, p.Val263Met and p.Gly362Val still have partial enzyme activity in vitro. This result is essentially consistent with the clinical manifestations of other patients reported to carry these mutations. However, the correlation between genotype and phenotype of type I citrullinemia has not been clarified so far. Some patients have been reported to develop severe hyperammonemia until they are in a hypercatabolic state (both during surgery and post-operative, post-partum, or fever).

Citrullinemia is a disease with chromosome abnormality, no radical treatment is available at present, low-protein diet and blood ammonia reducing treatment are mainly used for treatment, and if symptoms are serious or blood ammonia is too high, treatment is carried out by means of blood or peritoneal dialysis. Therefore, the medicine for treating citrullinemia has very important clinical value.

Tumor cells proliferate rapidly and require more energy and nutrients including nucleotides, proteins, lipids, etc. CAD [ Carbamoylphosphate synthase II (CPSase), Aspartate transcarbamylase (ATPase) and dihydroorotate dehydrogenase (DHOase) ] are key rate-limiting enzymes for de novo pyrimidine nucleotide synthesis. De novo biosynthesis of pyrimidine nucleotides is as follows: glutamine and carbon dioxide are supplied with energy by ATP in cytosol, and carbamyl phosphate synthetase II catalyzes to generate carbamyl phosphate. The latter in turn transfers a carbamoyl group to an amino group of aspartic acid to form carbamoylaspartic acid under the catalysis of aspartate transcarbamylase. The carbamyl aspartic acid is dehydrated and cyclized to generate dihydroorotic acid, and then the dihydroorotic acid is dehydrogenated to obtain the orotic acid. Orotic acid is an essential precursor of pyrimidine nucleotides. It follows that aspartic acid is also a key substrate for CAD. If the tumor cells proliferate abnormally, more aspartic acid must be involved in the synthesis of nucleotides.

Aspartic acid contains two carboxyl groups, has high polarity, and exogenous aspartic acid such as food and the like can hardly enter cells, and the source of the intracellular aspartic acid depends on endogenous biosynthesis. The catabolic pathway of aspartate determines its role and function within the cell. Aspartic acid is a common substrate for ASS1 and CAD, therefore, both ASS1 and CAD compete for the use of aspartic acid.

If the tumor cell ASS1 is down-regulated or defective, this results in increased use of its substrate aspartate for pyrimidine nucleotide synthesis, promoting cell proliferation. ASS1 is closely associated with tumor growth, and in some tumors, ASS1 expression is down-regulated or defective, including breast, melanoma, hepatocellular, prostate, bladder, mesothelioma, ovarian, renal, pancreatic malignancies, nasopharyngeal, osteosarcoma, and myxofibrosarcoma, among others. There is a clear correlation between the deficiency of ASS1 and poor prognosis of cancer, which is considered as a tumor suppressor protein, suggesting that ASS1 exhibits a tumor suppressor function in various tumors, and particularly in tumors deficient in ASS1, activation of ASS1 can inhibit synthesis of pyrimidine nucleotides necessary for tumor cell proliferation via the aspartate pathway. Therefore, the ASS1 protein is considered to be a potential direct action target of anti-tumor drugs. To date, no literature has disclosed chemical small molecules that can modulate the activity of ASS 1.

Spinosyn, an intracellular secondary metabolite produced by aerobic fermentation of Saccharopolyspora spinosa (Saccharopolyspora spinosa), is a macrolide antibiotic and has pesticidal activity. Spinosyns are sold under the trade name Spinosad (SP), and the main active ingredients of the spinosyns are A (Spinosyn A, SPA, 85-90%) and D (Spinosyn D, 10-15%), and the Spinosyn comprises a unique tetracyclic structure and is connected with two different hexahydric sugars. SP is used as a broad-spectrum biological pesticide and mainly used for preventing and controlling Lepidoptera and Thysanoptera pests. The chronic toxicity test of the mammal shows that SP has no carcinogenicity, teratogenicity, mutagenicity or neurotoxicity, and the acute toxicity test shows that the toxicity to the mammal is low (the mouse orally takes (mg/kg) LD50 ═ 3783-.

The invention discloses spinosyn derivatives as ASS1 activators, which can activate ASS1 and mutant ASS1^G362VEnzyme activity for the treatment of ASS1 deficient phaseThe related diseases are especially citrullinemia and antitumor.

Disclosure of Invention

The invention aims to provide a new application of a compound, which belongs to a spinosad derivative and can be used as an arginine-substituted succinate synthetase (ASS1) activator.

The technical scheme of the invention is to provide a spinosyn derivative and application of medically acceptable salt thereof as an argininosuccinate synthetase (ASS1) activator, wherein the spinosyn derivative has a structural general formula (I):

wherein R1 is selected from the following groups II-VIII:

r8 and R9 are each independently selected from the group consisting of hydrogen, alkyl of 1 to 20 carbon atoms (preferably alkyl of 2 to 16 carbon atoms, more preferably alkyl of 2 to 10 carbon atoms), haloalkyl of 1 to 20 carbons (preferably haloalkyl of 2 to 16 carbon atoms, more preferably haloalkyl of 2 to 10 carbon atoms), alkyl of 1 to 10 carbon atoms substituted with 1 to 6 carbon alkylamino groups (preferably alkyl of 2 to 6 carbon atoms), hydroxyalkyl of 1 to 10 carbon atoms substituted with acyloxy (preferably hydroxyalkyl of 2 to 6 carbon atoms), arylmethyl, phosphoryl, alkanoyl of 1 to 10 carbon atoms (preferably alkanoyl of 2 to 6 carbon atoms), aroyl, alkanoyl of 1 to 10 carbon atoms, and aroyl,

Wherein J is selected from a halogen atom, R19R20N-, tetrahydropyrrolyl, piperidinyl, morpholinyl, piperazinyl,

Wherein R16 is selected from hydrogen, alkyl of 1-10 carbons (preferably)Alkyl of 1 to 6 carbon atoms);

r10, R11 and R12 are each independently selected from hydrogen, alkyl of 1 to 20 carbons (preferably alkyl of 2 to 16 carbon atoms), alkanyl of 1 to 20 carbons (preferably alkanyl of 2 to 16 carbon atoms, more preferably alkanyl of 2 to 10 carbon atoms), arylmethyl;

r13 is selected from hydrogen, R14R15N-, nitrogen-containing heterocycle, oxygen-containing heterocycle, sulfur-containing heterocycle, phosphorus-containing heterocycle;

r14, R15, R19 and R20 are all independently selected from hydrogen, alkyl with 1-6 carbon atoms and alkyl with 1-10 carbon atoms substituted by amino;

r2 is selected from ethyl, propyl, butyl, alkenyl of 3-4 carbons;

r3 is selected from hydrogen, methyl;

r4 is selected from hydrogen, hydroxylamino, -S-R17; wherein R17 is selected from hydrogen, C1-C6 substituted alkyl, C1-C6 alkenyl, arylmethyl, aryl, - (CH)₂)qCH₂YR 18; in- (CH)₂)qCH₂In YR18, R18 is selected from H, 1-6 carbon alkyl, aroyl, substituted aroyl, arylcarbamoyl, heteroaromatic acyl, 1-5 carbon alkyl acyl, aryl alkanoyl, N-substituted carbamoyl, alkoxyformyl, Y is oxygen or nitrogen atom, -q ═ 1,2 or 3;

r5, R6 and R7 are all independently selected from hydrogen, alkyl with 1-3 carbons, acetyl and propionyl;

r21 is selected from

A-B is selected from CH₂-CH₂、CH＝CH；

M-Q is selected from CH-CH, C ═ CH;

w is selected from CH₂、O、NH、S；

X is an anion;

x is anion, chlorine, bromine, iodine, sulfate radical, hydrogen sulfate radical, phosphate radical, methanesulfonic acid radical, benzenesulfonic acid radical, p-toluenesulfonic acid radical and hydroxyl radical;

n is an integer of 0 to 4, and m is an integer of 0 to 20.

Further, R2 is ethyl.

Further, R4 is hydrogen.

Further, R5, R6 and R7 are all independently selected from methyl or ethyl.

Further, W is selected from O, NH, NCH₃、S。

Further, the nitrogen-containing heterocycle, the oxygen-containing heterocycle, the sulfur-containing heterocycle and the phosphorus-containing heterocycle respectively mean that heteroatoms in the heterocycles are respectively nitrogen, oxygen, sulfur and phosphorus.

Further, the heteroatoms in the nitrogen-containing heterocycle are nitrogen atoms and the number of the heteroatoms is 1-3.

Further, the nitrogen-containing heterocycle is tetrahydropyrrolyl, piperidyl, morpholinyl, piperazinyl,

Wherein R16 is selected from alkyl of 1-10 carbons.

Further, the spinosyn derivative has the structure:

the invention relates to a compound shown in a general formula (I), which is characterized in that spinosad and derivatives thereof related to patents CN201610355188.0, ZL201010123056.8, CN201610356840.0 and US 6001981.

In addition, the invention also provides application of the small molecule composition as the argininosuccinate synthetase, wherein the small molecule composition comprises the compound shown in the structural general formula (I), and can also comprise one or more pharmaceutically acceptable carriers or excipients

The pharmaceutical composition comprises spinosad shown in a structural general formula (I) and derivatives thereof, and can also comprise one or more pharmaceutically acceptable carriers or excipients.

The carrier or excipient may comprise glycerol, ethanol, buffered saline, physiological saline, and combinations thereof. The pharmaceutical composition may further comprise a penetration enhancer, an antioxidant, and the like.

On the other hand, the invention provides application of spinosad shown as the general formula (I) and a derivative LM-2I thereof in preventing and treating diseases related to ASS1 low-expression or mutant ASS1G 362V. Wherein the ASS1 low expression or mutant ASS1G362V related diseases include but are not limited to type I citrullinemia and related cancers.

The invention identifies the small molecular target by using a biotin probe method, and discovers that the compound with the general structural formula (I) has the action target of ASS 1. The inventor synthesizes and discovers a spinosad A-biotin probe, such as Xn-03-17A (abbreviated as 17A), and finds that the spinosad A-biotin probe has good anti-tumor proliferation activity and can replace drugs to carry out target experiments. We use Xn-03-17A to carry out target point identification, and the result shows that the spinosyn derivative has the action target point of protein ASS 1. And Western blot analysis was performed using the ASS1 monoclonal antibody and confirmed to be ASS1 in combination with mass spectrometry.

The activation activity of spinosyn derivatives on ASS1 was determined according to the principle and method of determination of the activity of argininosuccinate synthetase (ASS 1). The principle of detecting the activity of ASS1 is as follows: ASS1 catalyzes the reaction of citrulline with aspartic acid to arginine-succinate, which consumes ATP to Produce Pyrophosphate (PPi), which produces phosphoric acid under the catalysis of pyrophosphatase, which reacts with ammonium molybdate to produce phosphomolybdic acid, which produces blue phosphomolybdic blue under the reduction of vitamin C, with a characteristic absorption peak at OD 660. The results show that spinosyn derivatives can increase the enzyme activity of ASS1 to different degrees, and the increase of the enzyme activity is 5.85-203.86% compared with the positive control of ASS 1.

The invention adopts MTT experiment to screen the activity of spinosad derivatives on various human tumor cells. The low expression of ASS1 is related to low overall survival rate and low disease-free survival rate of breast cancer patients, and ASS1 is an independent prognostic factor of the overall survival rate and the disease-free survival rate of the breast cancer and can be used as a novel candidate molecular typing marker of the breast cancer.

In-vitro enzyme activity experiments of ASS1 show that spinosad and derivatives LM-2I thereof can increase ASS1 to different degrees^G362VAn enzyme activity.

The invention has the beneficial effects that: spinosad and derivatives thereof are mutant argininosuccinate synthetase ASS1 which is discovered for the first time and targets oncostatin argininosuccinate synthetase 1(ASS1) and type I citrullinemia^G362VBecause ASS1 is generally expressed and down-regulated or has defects in tumors, spinosad and derivatives thereof have remarkable wide anti-tumor curative effects. At the same time, for ASS1 caused by mutation of ASS1 gene^G362VThe resulting congenital genetic disease difficult to cure, citrullinemia type I, spinosad and its derivatives can also be used as an orphan drug to reverse and recover ASS1^G362VThe functional activity exerts obvious curative effect. Spinosad and derivatives thereof are mainly used as drugs for treating diseases related to ASS1 deficiency, and are particularly used for type I citrullinemia and anti-tumor treatment.

Drawings

FIG. 1 shows a general structural formula of a spinosyn derivative;

FIG. 2 shows the reaction process catalyzed by argininosuccinate synthase 1(ASS 1);

FIG. 3 shows the aspartate metabolic pathway;

FIG. 4 shows the identification of the target of action of a compound; in the figure, 1A) silver staining results of Pull-down products in cell lysates; 1B) ASS1 antibody Western blot detection map;

FIG. 5 shows the mass spectrometry identification of the protein to which the probe Biotin-SPA binds as ASS 1;

FIG. 6 shows the relative catalytic activity of different compounds on ASS 1;

FIG. 7 shows the effect of spinosyn A (SPA) and LM-2I on the activity of mutant ASS 1;

FIG. 8 shows ASS1 and ASS1^G362VIn vitro enzyme of (2)The experimental results are alive;

FIG. 9 shows that there is a difference in the expression of ASS1 in breast cancer multi-cell lines. (a) Western blot for detecting the expression of ASS1 in various breast cancer cell lines, performing grayscale scanning on a band by software Image J, performing relative quantification by taking MDA-MB-231 with the lowest expression level as a reference and defining the band as 1, and obtaining a relative expression profile (b) of ASS 1;

FIG. 10 shows that the proliferation rate of breast cancer multi-cell lines is significantly inversely correlated with the expression level of ASS 1. MTT method for detecting proliferation rate (a) of the multi-cell line 48h, and (b) correlation analysis between the cell proliferation rate and the expression level of ASS 1;

FIG. 11 shows the detection of drug susceptibility following knockdown or overexpression of ASS 1. SPA or LM-2I treated MDA-MB-231 ASS1 overexpression cell line (a) or MCF-7ASS1 knockdown cell line (b) for 48h, respectively, and MTT detected cell survival. Each set of experiments was repeated three times (mean ± s.d.; n ═ 3), representing p < 0.001.

FIG. 12 shows that SPA and LM-2I inhibit the increase in tumor volume. SPA (10mg/kg/d) and LM-2I (5m/kg/d) were administered for 18 days, every other day, and the longest diameter and the shortest diameter of tumor bodies were recorded, and the tumor body volume was calculated by a formula, wherein Vehicle is a solvent control, and the experimental results were counted by mean + -s.d., n is 7, p is < 0.05, and p is < 0.01.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

Target identification and confirmation experiments

The specific test steps are as follows:

1) synthesis of a Biotin-based Spinosad Probe Spinosad-Biotin Co-Compound Xn-03-17A.

(1) Synthesis of N- (3-bromohexylalkyl) spinosad A100 mL single-neck round-bottom flask was charged with spinosad B1 g (1.39mmoL), acetonitrile 20mL and 385mg (1.39mmoL) of potassium carbonate, and finally 1, 6-dibromohexane 2.14 mL (13.9mmoL) was added, stirred at 45 ℃ for 48 hours, the resulting solid was removed by suction filtration, the solvent was removed, extracted with 50mL of water and EA (3 × 60mL), the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed, and then the mixture was passed through a silica gel column layer V (acetic acid)Ethyl ester): v (petroleum ether) ═ 4:1 gave 840mg of white solid, 73% yield. m.p.95-98 ℃; HRMS calcd. for₄₆H₇₄BrNO₁₀880.4574，found 880.4602。

(2) Synthesis of spinosyn-hexy-biotin (Xn-03-17A): to a 50mL single neck round bottom flask was added 1.5mL of DMF, 5100mg (0.11mmoL) of Compound, and 31mg (0.13mmoL) of biotin, and finally K was added₂CO₃16mg (0.11mmoL), stirring at room temperature for 12h, adding 15mL of water to the reaction solution, precipitating a large amount of white precipitate, extracting with EA (3 × 25mL), combining organic phases, drying with anhydrous sodium sulfate, removing the solvent, purifying by silica gel column chromatography V (ethyl acetate): V (methanol): 20:1, recrystallizing with V (n-hexane): V (isopropanol): 6:1 to obtain 68mg of white powder with a yield of 57%, m.p.124-129 ℃;¹HNMR(500MHz,CDCl₃):6.78(s,1H),5.90(d,J＝9.7Hz,1H),5.82 (m,1H),5.22(s,1H),4.97(s,1H),4.87(d,J＝1.3Hz,1H),4.73-4.64(m,1H),4.57-4.50(m,1H), 4.44(d,J＝6.6Hz,1H),4.38-4.28(m,2H),4.08(t,J＝6.7Hz,2H),3.68-3.61(m,1H),3.59-3.56(m,4H),3.54-3.46(m,10H),3.33-3.27(m,1H),3.20-3.11(m,3H),3.06-2.99(m,1H),2.96-2.92 (m,1H),2.91-2.86(m,1H),2.77(d,J＝12.8Hz,1H),2.43-2.33(m,5H),2.30-2.25(m,2H),2.19(s, 3H),1.99-1.92(m,2H),1.74-1.61(m,10H),1.59-1.53(m,4H),1.52-1.45(m,6H),1.39-1.32(m, 6H),1.30(d,J＝6.2Hz,4H),1.26-1.25(m,3H),1.20(d,J＝6.8Hz,4H),0.95-0.88(m,1H),0.84(t, J＝7.5Hz,3H)；HRMS calcd.forC₅₆H₈₉N₃O₁₃S 1044.6194，found1044.6234。

2) probe Xn-03-17A seeks interacting proteins (pull-down experiment).

The method comprises the steps of incubating Xn-03-17A and MCF-7 cell lysate for 4 ℃ overnight, adding streptavidin magnetic beads for incubation for 4 hours at room temperature, setting drug competition groups of 10 times and 20 times, adsorbing the magnetic beads by a magnetic field, repeatedly washing the magnetic beads for 4 times by using IP lysate, adding SDS-loading buffer for boiling for 10 minutes, staining with silver nitrate after SDS-PAGE, and independently repeating the experiment for three times to obtain the same result (n is 3). As shown in FIG. 4, the results show that the Xn-03-17A lane has a specific binding band, whereas the 10-fold and 20-fold drug groups can compete without a band. MS identification is carried out on the specific bands, and the result shows that the action target of the spinosad and derivatives thereof is protein ASS 1. Wherein FIG. 4A is a graph showing the results of silver staining of Pull-down products from MCF-7 cell lysates using Xn-03-17A probe. FIG. 4B is a Western blot assay using ASS1 antibody. The invention confirms that ASS1 is an action target of spinosyn derivatives.

3) Subsequently, we cut the specific band, perform MS/MS mass spectrometry after in-gel digestion, find that the confidence level is ASS1 by comparing the protein database, we detect the specific peptide fragment and the specific map together, cover 117 amino acids, ASS1 contains 412 amino acids in total, the detected peptide fragment accounts for 28.4%, and the molecular weight is 47kDa (fig. 3), which matches the silver staining result. In order to confirm the mass spectrum result, we used ASS1 specific monoclonal antibody to perform Western blot detection on the Pull-down product, cut the specific band obtained in fig. 3, perform mass spectrum analysis after enzymolysis, and compare the database to find that the peptide segment identified by ASS1 is the most and the score is the highest, and the red mark in the figure represents the identified peptide segment (fig. 5), and the result shows that the dragged protein is indeed ASS 1.

Example 2

Effect of drugs on the enzymatic Activity of protein ASS1

The principle of detecting the activity of ASS1 is as follows: ASS1 catalyzes the reaction of citrulline with aspartic acid to arginine-substituted succinic acid, which consumes ATP to produce pyrophosphoric acid (PPi), which produces phosphoric acid under the catalysis of pyrophosphatase, which reacts with ammonium molybdate to produce phosphomolybdic acid, which produces blue phosphomolybdic blue under the reduction of vitamin C, with a characteristic absorption peak at OD 660.

Enzyme activity calculation formula:

the enzyme activity is as follows: [ (drug + ASS1) OD₆₆₀-NCOD₆₆₀]/(ASS1OD₆₆₀-NCOD₆₆₀)×100％。

(drug + ASS1) OD₆₆₀: absorbance of the solution (660 nm wavelength) with drug and ASS1 added; ASS1OD₆₆₀:

Absorbance of solution at addition of ASS1 (wavelength 660 nm); NCOD₆₆₀: absorbance (wavelength 660nm) of blank reference solution.

1) Prokaryotic expression of ASS1

(1) Construction of prokaryotic expression vector

Extracting total RNA of a human breast cancer cell line MCF-7, amplifying ASS1 gene by RT-PCR, and using primers (5 '-3'):

ASS1-F：ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC(SEQ ID NO 3)；

ASS1-R：AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT

(SEQ ID NO 4)。

extracting pET-28a plasmid, digesting, recovering gel, and integrating ASS1 gene into pET-28a plasmid by homologous recombination.

And (3) transformation: and (3) transforming the constructed pET-28a plasmid into escherichia coli DH5a, screening positive clones for sequencing, carrying out amplification culture on the clones with correct sequencing, extracting the plasmids, transforming the plasmids into a BL21(DE3) strain, carrying out PCR (polymerase chain reaction) identification on the positive clones, determining the size of a strip to be 1.0-1.5kb, and carrying out sequencing. Selecting strains with correct sequencing, and preserving at-80 ℃ for later use.

(2) Prokaryotic expression: taking out constructed ASS1 BL21(DE3) strain stored at-80 deg.C, restoring and culturing to OD 660-0.4-0.6, adding 1mM IPTG to induce for 4h, centrifuging, collecting bacterial liquid, and storing at-80 deg.C for use. And (3) protein extraction: thawing a bacterial solution stored at the temperature of minus 80 ℃ on ice, adding lysine buffer flushing liquid according to 2-5mL per gram for resuspending bacteria, adding lysozyme until the final concentration is 1mg/mL, standing on ice for 30min, ultrasonically breaking the wall, centrifuging, taking the supernatant, and standing on ice for later use.

(3) And (3) protein purification: adding 1mL of 50% Ni-NTA filler into every 4mL of lysate, performing rotary incubation at 4 ℃ for 60min, loading the mixture into a column, washing twice with 1mL of the filler each time, washing 4 times with the elusion buffer with 1mL of the filler each time, and collecting the liquid eluted four times.

(4) Desalting and preserving of proteins: placing the eluate in an ultrafiltration tube, centrifuging to remove salt, adjusting the storage buffer according to the nature of the protein, and adjusting the volume of the buffer according to the desired concentration.

(5) Subpackaging and storing: subpackaging the protein with determined protein concentration, subpackaging 10-20 μ L each, quick freezing with liquid nitrogen, and storing at-80 deg.C.

2)0.5 μ M ASS1 was incubated with 20 μ M drug overnight at 4 deg.C, buffer was added and incubated for 1min at 37 deg.C, after which an equal volume of Molybdate buffer was added and developed for 3min at 37 deg.C and the absorbance was measured at OD 660. ASS1 group without drug incubation served as positive control, and the reagent group without ASS 1as NC. The buffer solution comprises the following components: 20mM Tris & HCl (pH 7.8), 2mM ATP, 10mM citrulline, 10mM aspartic acid, 6mM MgCl2, 20mM KCl, 0.2U pyrophosphatase. The Molybdate buffer component is as follows: vitamin C10 mM, ammonium molybdate 2.5mM, sulfuric acid 2% (V/V) (results of enzyme activation FIG. 6 and Table 1).

TABLE 1 relative Activity of spinosyn derivatives to activate argininosuccinate synthetase

The results show that the spinosyn derivatives can increase the enzyme activity of ASS1 to different degrees, and the increase of the enzyme activity is 5.8-203.9% compared with the positive control of ASS 1. Half activation concentration AC of SPA and LM-2I₅₀Respectively 18.6 and 2.0 μm.

Example 3

Drug-pair proteins ASS1 and ASS1^G362VInfluence of enzyme Activity

For detecting SPA and its derivative LM-2I on proteins ASS1 and ASS1^G362VUnder the influence of enzyme activity, ASS1 pET28a plasmid is constructed by homologous recombination technology, and ASS1 is constructed by adopting gene site-directed mutagenesis technology on the basis of the plasmid^G362VpET28a plasmid, transferring the constructed plasmid into BL21(DE3) for prokaryotic expression and purifying proteins ASS1 and ASS1^G362V。

The method comprises the following specific steps:

3.1ASS1 and ASS1^G362VAmino acid sequence

ASS1 has the amino acid sequence:

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMSSKGSVVLAYSGGLDTSCIL VWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALY EDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAP WRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPP GLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGR IDIVENRFIGMKSRGIYETPAGTILYHAHLDIEAFTMDREVRKIKQGLGLKFAELVYTGFWHS PECEFVRHCIAKSQERVEGKVQVSVLKGQVYILGRESPLSLYNEELVSMNVQGDYEPTDATGFININSLRLKEYHRLQSKVTAK(SEQ ID NO 1)。

ASS1^G362Vamino acid sequence:

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMSSKGSVVLAYSGGLDTSCIL VWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALY EDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAP WRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPP GLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGR IDIVENRFIGMKSRGIYETPAGTILYHAHLDIEAFTMDREVRKIKQGLGLKFAELVYTGFWHS PECEFVRHCIAKSQERVEGKVQVSVLKGQVYILVRESPLSLYNEELVSMNVQGDYEPTDATGFININSLRLKEYHRLQSKVTAK(SEQ ID NO 2)。

ASS1G at position 362, ASS1G^362VAt position 362 is a V in parentheses.

3.2ASS1-pET-28a plasmid construction

(1) Extracting total RNA of a human breast cancer cell line HCC1806 cell line, amplifying ASS1 gene by RT-PCR, and using primers (5 '-3'):

ASS1-F：ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC(SEQ ID NO 3)；

ASS1-R：AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT(SEQ ID NO 4)。

(2) extracting pET-28a plasmid, recovering restriction enzyme glue, and integrating ASS1 gene into pET-28a plasmid according to a homologous recombination method.

(3) And (3) transformation: the constructed pET-28a plasmid is transformed into Escherichia coli DH₅In a, screening positive clones, sequencing, carrying out amplification culture on the clones with correct sequencing, extracting plasmids, and transforming the plasmids into BL21 (DE)₃) In the strain, positive clones were identified by PCR, the size of the band was determined to be between 1.0-1.5kb, and sequencing was performed.

Wherein the sequence of the ASS1 variant 1 (ACCESSION: NM-000050) in the NCBI database of the nucleotide sequence of ASS1 is completely consistent with the sequence of the ASS1 in the Uniprot database when translated into amino acids, so the constructed ASS1 prokaryotic expression vector can be used for subsequent research. Selecting strains with correct sequencing, and preserving at-80 ℃ for later use.

3.3ASS1^G362VConstruction of expression plasmid for-pET-28 a

The Mut Express II Fast Mutagenesis kit V2 kit from Nanjing Novowed Biotechnology Inc. was used, specifically as follows:

designing a point mutation primer, and designing the mutation primer according to the principle of homologous recombination:

ASS1^G362V-F：TACATCCTCGTCCGGGAGTCCCCACTGTCTCTCTACAAT

(SEQ ID NO 5)

ASS1^G362V-R：GGACTCCCGGACGAGGATGTACACCTGGCCCTTGAGGAC

(SEQ ID NO 6)

and (3) PCR amplification: amplifying pet28a-ASS1 plasmid by using a mutation primer; dpnl digestion of the amplified product to remove the methylated template plasmid; carrying out homologous recombination reaction, transformation, sequencing and identification.

3.4 expression and purification of proteins

(1) Prokaryotic expression: removal of well-constructed ASS1 BL21 (DE)₃) The strain is recovered and cultured to OD₆₆₀Adding IPTG with final concentration of 1mM for induction for 4h when the temperature is between 0.4 and 0.6, centrifugally collecting bacterial liquid, and storing at-80 ℃ for later use.

(2) And (3) protein extraction: thawing the bacteria liquid stored at-80 ℃ on ice, adding lysine buffer flushing liquid according to 2-5mL per gram for resuspending bacteria, adding lysozyme to the final concentration of 1mg/mL, standing on ice for 30min, ultrasonically breaking the wall, centrifuging, taking the supernatant, and standing on ice for later use.

(3) And (3) protein purification: adding a filler according to the proportion that 1mL of 50% Ni-NTA filler is added into every 4mL of lysate, carrying out rotary incubation at 4 ℃ for 60min, loading the mixture into a column, washing twice by wash buffer, 1mL each time, washing 4 times by the elusion buffer, 1mL each time, and collecting the liquid eluted four times.

Wherein: lysine buffer (1L) (protease inhibitor added before use, 5mM mercaptoethanol added before use): HCl 50mM Tris, 500mM NaCl, 10mM imidozole, Adjust pH to 8.0using NaOH;

wash buffer (1L) (without protease inhibitor, 5mM mercaptoethanol before use): 50mM Tris.HCl, 500mM NaCl, 20mM imidazole, Adjust pH to 8.0using NaOH;

elution buffer (1L) (without protease inhibitor, without 5mM mercaptoethanol): 50mM Tris.HCl, 500mM NaCl, 250mM imidazole, Adjust pH to 8.0using NaOH

After elution, desalting was replaced (20mM Tris.HCl + 100-.

Detection of ASS1 and ASS1 after purification by Coomassie brilliant blue staining^G362VExpression purification (figure 7). Results display ASS1 and ASS1^G362VBoth proteins were expressed in supernatant, dissolved in water and purified to over 90% purity.

3.5 in vitro enzyme Activity test

ASS1 and ASS1^G362VThe concentration is 0.5 μ M, the concentration is 10 μ M with spinosad and spinosad derivative LM-2I, the mixture is incubated at 4 ℃ overnight, buffer solution is added, the mixture is incubated at 37 ℃ for 1min, then equal volume of Molybdate buffer is added for developing for 3min, the OD is at 37 ℃ and₆₆₀and detecting the absorbance. ASS1 group without drug incubation served as positive control, and the reagent group without ASS 1as NC.

The relative enzyme activities were: [ ASS1^G362VOD₆₆₀-NCOD₆₆₀]/(ASS1OD₆₆₀-NCOD₆₆₀)×100％。

Wherein the buffer solution comprises the following components: 20mM Tris. HCl (pH 7.8), 2mM ATP, 10mM citrulline, 10mM aspartic acid, 6mM MgCl2, 20mM KCl, 0.2U pyrophosphatase. The Molybdate buffer component is as follows: 10mM vitamin C, 2.5mM ammonium molybdate, 2% (V/V) sulfuric acid.

Through an ASS1 in vitro enzyme activity experiment, SPA and LM-2I can restore the activity of mutant ASS 1. ASS1 and ASS1^G362VThe reaction concentration is 0.5 mu M, the concentration LM-2I of spinosad A (SPA) and derivatives thereof is 10 mu M, the reaction time is 1min, the single ASS1 is a positive control, and the result shows that the spinosad and derivatives thereof LM-2I can increase the ASS1 to different degrees^G362VEnzyme activity, wherein ASS1 is positive control, and its enzyme activity is normalized to 100 + -6.49%, ASS1^G362VThe enzyme activity was 60.43 + -3.64%, 10 μ M SPA-treated ASS1^G362VThe post-enzyme activity was 70.8. + -. 4.22% with ASS1^G362VCompared with the enzyme activity, the activity is increased by 10.37 percent and is not obvious; significantly reduced compared to ASS1 enzyme activity. 10 μ M LM-2I Process ASS1^G362VPost-enzyme activity 94.12. + -. 1.83% with ASS1^G362VCompared with the enzyme activity, the activity is increased by 33.69 percent and is obviously increased; substantially in agreement with ASS1 enzyme activity (fig. 8).

Example 4

Effect of Compounds on Activity of tumor cells at different ASS1 expression levels

1 construction of lentivirus overexpression vector and shRNA interference vector

The overexpression vector and shRNA interference vector of ASS1 were purchased from the gimeram gene, and its shRNA has two sequences (5 '-3'):

ASS1 shRNA1：

TGGATGTCAGCAGGGAGTTTGTTTCAAGAGAACAAACTCCCTGCTGACATCCTTTTTTC( SEQ IDNO 7)。

ASS1 shRNA2：

TGGAGGATGCCTGAATTCTACATTCAAGAGATGTAGAATTCAGGCATCCTCCTTTTTTC(S EQ IDNO 8)。

The negative control shRNAsequence：

TGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTC(SE Q ID NO9)。

the ASS1 overexpression sequence was obtained by total synthesis, and the sequence was the CDS sequence of human ASS1 variant 1 (ACCESSION: NM-000050).

2 packaging of lentiviruses

1) Recovering 293T tool cells to be cultured to be in a good state, digesting the cells by trypsin, inoculating the cells into a 10cm culture dish, culturing the cells overnight, and packaging lentiviruses when the cells grow to be 90% fused.

2) Taking two 1.5mL sterile centrifuge tubes, adding 600 mu L serum-free DMEM medium into one tube, proportionally adding the target plasmid and the packaging plasmid (pGag/Pol, pRev and pVSV-G), mixing uniformly, adding 600 mu L serum-free DMEM medium into the other tube, adding 120 mu L RNAi-Mate, mixing uniformly, standing at room temperature for 5min, mixing uniformly, and standing at room temperature for 20 min.

3) And (3) replacing the complete culture medium in the culture dish with a serum-free DMEM culture medium, adding the prepared transfection system into the culture dish drop by drop, slightly shaking and uniformly mixing all the materials, and continuously culturing in the incubator.

4) And after continuing to culture for 6h, removing the serum-free DMEM medium, replacing with a complete medium, and continuing to culture for 72 h.

5) Collecting supernatant in the culture dish to a 50mL centrifuge tube to obtain virus stock solution, centrifuging the virus stock solution at low temperature and low speed at 4 deg.C and 4000rpm for 15min, and filtering the supernatant with 0.45 μm filter.

6) And (3) performing ultracentrifugation on the filtered virus liquid at 4 ℃ and 20000rpm for 2h, removing supernatant, adding a proper amount of culture medium to dissolve viruses, subpackaging the viruses into 1.5mL centrifuge tubes, labeling and storing at-80 ℃.

3 Lentiviral titer detection (GFP fluorescence counting method)

1)293T tool cells were cultured to 90% confluence, trypsinized, centrifuged, resuspended, and counted.

2) Cells were seeded at 3X 104 cells/well in 96-well plates and cultured for 24h after mixing.

3) mu.L of the lentivirus stock solution was diluted 10-fold with complete medium in 5 gradients and Polybrene was added to a final concentration of 5. mu.g/mL to increase the infection efficiency.

4) Removing the original culture medium in the 96-well plate, adding virus culture medium with different concentration gradients, continuously culturing for 24h, and setting blank control.

5) Removing the virus-containing culture medium, and culturing for 72h with complete culture medium.

6) GFP fluorescence was observed with a fluorescence microscope, fluorescent cells were counted, and virus titer was calculated from the dilution factor.

4 Lentiviral infection and monoclonal screening

1) Recovering MDA-MB-231 and MCF-7 cells, culturing in 10cm culture dish to obtain good state, digesting, centrifuging and counting when the cells grow to 90% and are fused, and collecting MDA-MB-231(1 × 10)⁶Vessel) and MCF-7(1.5 × 10)⁶/dish) passage, and carrying out a lentivirus infection experiment when the cell density reaches 40-50%.

2) Based on the number of cells, the amount of virus required (MOI values of both cells are 50) and the volume of virus required were calculated, Polybrene was added to a final concentration of 5. mu.g/mL, the volume was made up to 8mL with complete medium, and mixed well.

3) Removing original culture medium of cells, adding culture medium containing virus, culturing for 24h, removing culture medium containing virus after 24h, and culturing for 72 h.

4) Infected cells are digested and passaged, Puromycin (Puromycin) with the final concentration of 2 mug/mL is added to screen positive cells, and the culture medium is kept to contain 2 mug/mL Puromycin during the subsequent culture process.

5) Surviving cells were digested, counted, seeded into 96-well plates at 0.5 cells per well, and single colony screened. And digesting the grown monoclonal pancreatin, inoculating the digested monoclonal pancreatin to a 6-well plate, and after the monoclonal pancreatin grows to about 80-90%, using half of the digested monoclonal pancreatin for passage and using half of the digested monoclonal pancreatin for extracting protein Western blot to detect the expression condition of the target protein. And carrying out amplification culture on the single clone meeting the requirement, storing the single clone by liquid nitrogen, and carrying out an MTT (methanol to transfer) experiment.

6) MTT method cell experiment, wherein the cell line knocked down or over-expressed by ASS1 is used for detecting the drug sensitivity of spinosad A (SPA) or LM-2I, and the SPA or LM-2I with the same concentration simultaneously treats MCF-7ASS1 sh and MCF-7NC or MDA-MB-231 ASS1 OE and MDA-MB-231NC cells for 48 h.

The results show that: as shown in FIG. 9a, ASS1 has large difference in expression level among different cell lines, the expression level is lowest in MDA-MB-231 and highest in MCF-7, for the convenience of statistics, we performed grayscale scanning on the bands using Image J, defined as 1 with MDA-MB-231 with the lowest expression level of ASS 1as a reference, and performed relative quantification to obtain ASS1 relative expression profile (FIG. 9b), with the value range of 1-11.16 + -0.57.

The relative proliferation rate of 48h of breast cancer cell lines was measured using the MTT method with 0h as reference, as shown in fig. 10, and we surprisingly found that: the relative expression level of ASS1 showed a significant negative correlation with the cell proliferation rate (Spearman's ρ -0.783, p-0.003) (fig. 10). The above results suggest that ASS1 may play a role as a tumor suppressor in breast cancer.

The results show that in the MDA-MB-231 cell line (FIG. 11), the same concentration of SPA or LM-2I treatment significantly increased the survival rate of ASS1 OE cells and decreased drug sensitivity compared to NC; in contrast, in the MCF-7 cell line (FIG. 11-b), ASS1 sh cells were significantly less viable and more sensitive to drugs than NC. Obviously, the spinosyn derivative has better sensitivity to the low-expression ASS1 tumor, and can be used as an individualized treatment drug for the low-expression ASS1 tumor. ASS1 low-expression tumor is not limited to breast cancer, such as melanoma, hepatocellular carcinoma, prostate cancer, bladder cancer, mesothelioma, ovarian cancer, renal cancer, pancreatic malignancy, nasopharyngeal carcinoma, osteosarcoma, and mucofibrosarcoma.

Example 5

In vivo tumor-inhibiting effect

A triple negative breast cancer MDA-MB-231 cell line which has low ASS1 expression level, high malignancy degree, easy transfer, poor prognosis and no targeted drug is selected as a representative for animal experiments.

4 weeks of birth immunodeficient nude mice BALB/c (nu/nu) were purchased and after one week of acclimation in SPF-grade animal housing, each nude mouse was injected with MDA-MB-231 cells 5 × 10⁶And 4-7 days later, the mouse grows into a tumor body with the size of rice grains, and the success of modeling is shown. The solvent group is a negative control group, the clinical breast cancer first-line treatment drug 5-FU is a positive control group, the treatment concentration is 10mg/kg/d, the SPA treatment group is 10mg/kg/d, the LM-2I treatment group is two groups, the concentrations are respectively 5mg/kg.d and 10 mg/kg.d, the drug administration is carried out every other day for 10 times, the living state of the mouse is observed every day, and the drug administration is carried out every timeWeights were weighed before dosing and tumor major a and minor b diameters were measured and tumor volumes were determined using the formula: v1/2 ab 2mm³。

18 days after administration, the tumor volume in the Vehicle group was 841.52. + -. 420.81mm³(ii) a SPA (10mg/kg/d) volume 386.27 + -77.06 mm³The reduction compared with Vehicle is 54.10%, and the statistical difference exists; the volume of the LM-2I (5mg/kg/d) group is 306.41 +/-79.08 mm³Compared with Vehicle, the reduction is 63.60%, and the difference is statistical. From the inhibitory effect, LM-2I (5mg/kg/d) was superior to SPA (10mg/kg/d), with the LM-2I drug concentration being only half of SPA (FIG. 12).

The experimental result proves that SPA and the derivative LM-2I can effectively inhibit the growth of ASS1 low-expression tumor. The spinosad and derivatives thereof can be used for individualized treatment of ASS1 low-expression and other deficient tumors.

SEQUENCE LISTING

<110> university of south-middle school

<120> spinosyn derivatives as activators of argininosuccinate synthetase and applications thereof

<130>1

<160>9

<170>PatentIn version 3.5

<210>1

<211>448

<212>PRT

<213> Artificial Synthesis

<400>1

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Met Ser Ser Lys Gly Ser Val Val Leu Ala Tyr Ser

35 40 45

Gly Gly Leu Asp Thr Ser Cys Ile Leu Val Trp Leu Lys Glu Gln Gly

50 55 60

Tyr Asp Val Ile Ala Tyr Leu Ala Asn Ile Gly Gln Lys Glu Asp Phe

6570 75 80

Glu Glu Ala Arg Lys Lys Ala Leu Lys Leu Gly Ala Lys Lys Val Phe

85 90 95

Ile Glu Asp Val Ser Arg Glu Phe Val Glu Glu Phe Ile Trp Pro Ala

100 105 110

Ile Gln Ser Ser Ala Leu Tyr Glu Asp Arg Tyr Leu Leu Gly Thr Ser

115 120 125

Leu Ala Arg Pro Cys Ile Ala Arg Lys Gln Val Glu Ile Ala Gln Arg

130 135 140

Glu Gly Ala Lys Tyr Val Ser His Gly Ala Thr Gly Lys Gly Asn Asp

145 150 155 160

Gln Val Arg Phe Glu Leu Ser Cys Tyr Ser Leu Ala Pro Gln Ile Lys

165 170 175

Val Ile Ala Pro Trp Arg Met Pro Glu Phe Tyr Asn Arg Phe Lys Gly

180 185 190

Arg Asn Asp Leu Met Glu Tyr Ala Lys Gln His Gly Ile Pro Ile Pro

195 200 205

Val Thr Pro Lys Asn Pro Trp Ser Met Asp Glu Asn Leu Met His Ile

210 215 220

Ser Tyr Glu Ala Gly Ile Leu Glu Asn Pro Lys Asn Gln Ala Pro Pro

225230 235 240

Gly Leu Tyr Thr Lys Thr Gln Asp Pro Ala Lys Ala Pro Asn Thr Pro

245 250 255

Asp Ile Leu Glu Ile Glu Phe Lys Lys Gly Val Pro Val Lys Val Thr

260 265 270

Asn Val Lys Asp Gly Thr Thr His Gln Thr Ser Leu Glu Leu Phe Met

275 280 285

Tyr Leu Asn Glu Val Ala Gly Lys His Gly Val Gly Arg Ile Asp Ile

290 295 300

Val Glu Asn Arg Phe Ile Gly Met Lys Ser Arg Gly Ile Tyr Glu Thr

305 310 315 320

Pro Ala Gly Thr Ile Leu Tyr His Ala His Leu Asp Ile Glu Ala Phe

325 330 335

Thr Met Asp Arg Glu Val Arg Lys Ile Lys Gln Gly Leu Gly Leu Lys

340 345 350

Phe Ala Glu Leu Val Tyr Thr Gly Phe Trp His Ser Pro Glu Cys Glu

355 360 365

Phe Val Arg His Cys Ile Ala Lys Ser Gln Glu Arg Val Glu Gly Lys

370 375 380

Val Gln Val Ser Val Leu Lys Gly Gln Val Tyr Ile Leu Gly Arg Glu

385 390395 400

Ser Pro Leu Ser Leu Tyr Asn Glu Glu Leu Val Ser Met Asn Val Gln

405 410 415

Gly Asp Tyr Glu Pro Thr Asp Ala Thr Gly Phe Ile Asn Ile Asn Ser

420 425 430

Leu Arg Leu Lys Glu Tyr His Arg Leu Gln Ser Lys Val Thr Ala Lys

435 440 445

<210>2

<211>448

<212>PRT

<213> Artificial Synthesis

<400>2

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Met Ser Ser Lys Gly Ser Val Val Leu Ala Tyr Ser

35 40 45

Gly Gly Leu Asp Thr Ser Cys Ile Leu Val Trp Leu Lys Glu Gln Gly

50 55 60

Tyr Asp Val Ile Ala Tyr Leu Ala Asn Ile Gly Gln Lys Glu Asp Phe

65 70 75 80

Glu Glu Ala Arg Lys Lys Ala Leu Lys Leu Gly Ala Lys Lys Val Phe

85 90 95

Ile Glu Asp Val Ser Arg Glu Phe Val Glu Glu Phe Ile Trp Pro Ala

100 105 110

Ile Gln Ser Ser Ala Leu Tyr Glu Asp Arg Tyr Leu Leu Gly Thr Ser

115 120 125

Leu Ala Arg Pro Cys Ile Ala Arg Lys Gln Val Glu Ile Ala Gln Arg

130 135 140

Glu Gly Ala Lys Tyr Val Ser His Gly Ala Thr Gly Lys Gly Asn Asp

145 150 155 160

Gln Val Arg Phe Glu Leu Ser Cys Tyr Ser Leu Ala Pro Gln Ile Lys

165 170 175

Val Ile Ala Pro Trp Arg Met Pro Glu Phe Tyr Asn Arg Phe Lys Gly

180 185 190

Arg Asn Asp Leu Met Glu Tyr Ala Lys Gln His Gly Ile Pro Ile Pro

195 200 205

Val Thr Pro Lys Asn Pro Trp Ser Met Asp Glu Asn Leu Met His Ile

210 215 220

Ser Tyr Glu Ala Gly Ile Leu Glu Asn Pro Lys Asn Gln Ala Pro Pro

225 230 235 240

Gly Leu Tyr Thr Lys Thr Gln Asp Pro Ala Lys Ala Pro AsnThr Pro

245 250 255

Asp Ile Leu Glu Ile Glu Phe Lys Lys Gly Val Pro Val Lys Val Thr

260 265 270

Asn Val Lys Asp Gly Thr Thr His Gln Thr Ser Leu Glu Leu Phe Met

275 280 285

Tyr Leu Asn Glu Val Ala Gly Lys His Gly Val Gly Arg Ile Asp Ile

290 295 300

Val Glu Asn Arg Phe Ile Gly Met Lys Ser Arg Gly Ile Tyr Glu Thr

305 310 315 320

Pro Ala Gly Thr Ile Leu Tyr His Ala His Leu Asp Ile Glu Ala Phe

325 330 335

Thr Met Asp Arg Glu Val Arg Lys Ile Lys Gln Gly Leu Gly Leu Lys

340 345 350

Phe Ala Glu Leu Val Tyr Thr Gly Phe Trp His Ser Pro Glu Cys Glu

355 360 365

Phe Val Arg His Cys Ile Ala Lys Ser Gln Glu Arg Val Glu Gly Lys

370 375 380

Val Gln Val Ser Val Leu Lys Gly Gln Val Tyr Ile Leu Val Arg Glu

385 390 395 400

Ser Pro Leu Ser Leu Tyr Asn Glu Glu Leu Val Ser Met Asn Val Gln

405 410 415

Gly Asp Tyr Glu Pro Thr Asp Ala Thr Gly Phe Ile Asn Ile Asn Ser

420 425 430

Leu Arg Leu Lys Glu Tyr His Arg Leu Gln Ser Lys Val Thr Ala Lys

435 440 445

<210>3

<211>39

<212>DNA

<213> Artificial Synthesis

<400>3

accctcgagg gatccgaatt catgtccagc aaaggctcc 39

<210>4

<211>39

<212>DNA

<213> Artificial Synthesis

<400>4

agactgcagg tcgacaagct tttatttggc agtgacctt 39

<210>5

<211>39

<212>DNA

<213> Artificial Synthesis

<400>5

tacatcctcg tccgggagtc cccactgtct ctctacaat 39

<210>6

<211>39

<212>DNA

<213> Artificial Synthesis

<400>6

ggactcccgg acgaggatgt acacctggcc cttgaggac 39

<210>7

<211>59

<212>DNA

<213> Artificial Synthesis

<400>7

tggatgtcag cagggagttt gtttcaagag aacaaactcc ctgctgacat ccttttttc 59

<210>8

<211>59

<212>DNA

<213> Artificial Synthesis

<400>8

tggaggatgc ctgaattcta cattcaagag atgtagaatt caggcatcct ccttttttc 59

<210>9

<211>57

<212>DNA

<213> Artificial Synthesis

<400>9

tgttctccga acgtgtcacg tttcaagaga acgtgacacg ttcggagaac ttttttc 57

Claims

1. Spinosyn derivatives and their medically acceptable salts as activators of argininosuccinate synthase, characterized in that the spinosyn derivatives have the general structural formula (I):

wherein R1 is selected from the following groups II-VIII:

r8 and R9 are independently selected from hydrogen, alkyl with 1-20 carbon atoms, halogenated alkyl with 1-20 carbon atoms, alkyl with 1-10 carbon atoms substituted by 1-6 carbon alkylamino groups, alkyl with 1-10 carbon atoms substituted by acyloxy groups, arylmethyl, phosphoryl, alkanoyl with 1-10 carbon atoms, aroyl, and the like,

Wherein R16 is selected from hydrogen, alkyl of 1-10 carbons;

r10, R11 and R12 are all independently selected from hydrogen, alkyl with 1-20 carbons, alkyl alkenyl with 1-20 carbons and arylmethyl;

r2 is selected from ethyl, propyl, butyl, alkenyl of 3-4 carbons;

r3 is selected from hydrogen, methyl;

r4 is selected from hydrogen, hydroxylamino, -S-R17(ii) a Wherein R17 is selected from hydrogen, C1-C6 substituted alkyl, C1-C6 alkenyl, arylmethyl, aryl, - (CH)₂)qCH₂YR 18; in- (CH)₂)qCH₂In YR18, R18 is selected from H, alkyl having 1 to 6 carbon atoms, aroyl, substituted aroyl, arylaminocarbonyl, heterocycloaromatic, alkyl acyl having 1 to 5 carbon atoms, arylalkanoyl, N-substituted carbamoyl, alkoxyformyl, Y is oxygen or nitrogen atom, q is 1,2 or 3;

r21 is selected from

A-B is selected from CH₂-CH₂、CH＝CH；

M-Q is selected from CH-CH, C ═ CH;

w is selected from CH₂、O、NH、NCH₃、S；

X is an anion; x is selected from chlorine, bromine, iodine, sulfate radical, hydrogen sulfate radical, phosphate radical, methanesulfonate radical, benzenesulfonate radical, p-toluenesulfonate radical and hydroxyl radical.

n is an integer of 0 to 4, m is an integer of 0 to 20, and k is an integer of 0 to 20.

2. The use of claim 1, wherein R2 is ethyl; r5, R6 and R7 are all independently selected from methyl or ethyl; w is selected from O, NH, NCH₃、S。

3. The use as claimed in claim 1, wherein the heteroatoms in the nitrogen-containing heterocycle are nitrogen atoms and are present in an amount of 1 to 3.

4. The use of claim 1, wherein the nitrogen-containing heterocycle is tetrahydropyrrolyl, piperidinyl, morpholinyl, piperazinyl,

Wherein R is16 is selected from alkyl groups of 1 to 10 carbons.

5. The use of claim 1 wherein the spinosyn derivative has the following structural formula:

6. use of a spinosyn derivative and pharmaceutically acceptable salts thereof for the use of any of claims 1-5 in the manufacture of a medicament for the treatment of a disease associated with argininosuccinate synthetase.

7. Use according to claim 6, characterized in that the argininosuccinic acid synthetase is in particular the proteins ASS1 and ASS1^G362V(ii) a The proteins ASS1 and ASS1^G362VThe preparation method comprises the following steps: ASS1 pet28a plasmid is constructed by homologous recombination technology, and then ASS1 is constructed by adopting gene site-directed mutagenesis technology^G362Vpet28a plasmid, and respectively transferring the two constructed plasmids into Escherichia coli DH₅a prokaryotic expression and purification of proteins ASS1 and ASS1^G362V。

8. The use according to claim 7, characterized in that the proteins ASS1 and ASS1^G362VThe preparation steps comprise:

construction of plasmids S1 and ASS1-pET-28a

ASS1-F：ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC(SEQ ID NO 3)；

ASS1-R：AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT(SEQ ID NO 4)；

(2) extracting pET-28a plasmid, carrying out enzyme digestion and electrophoretic gel recovery, and then integrating the ASS1 gene into the pET-28a plasmid according to a homologous recombination method;

(3) and (3) transformation: the constructed pET-28a plasmid is transformed into Escherichia coli DH₅Screening positive clones, sequencing, carrying out amplification culture on the clones with correct sequencing, extracting plasmids, transforming the plasmids into a BL21 strain, carrying out PCR (polymerase chain reaction) to identify the positive clones, determining the size of a band to be 1.0-1.5kb, and sequencing; selecting strains with correct sequencing, and preserving the strains at-80 ℃ for later use;

S2、ASS1^G362Vconstruction of expression plasmid for-pET-28 a

ASS1^G362V-F：TACATCCTCGTCCGGGAGTCCCCACTGTCTCTCTACAAT(SEQ ID NO 5)

ASS1^G362V-R：GGACTCCCGGACGAGGATGTACACCTGGCCCTTGAGGAC(SEQ ID NO 6)

and (3) PCR amplification: amplifying the pET28a-ASS1 plasmid by using a mutation primer; dpnl digestion of the amplified product to remove the methylated template plasmid; carrying out homologous recombination reaction, transformation and sequencing identification;

s3, expression and purification of protein

(1) Prokaryotic expression: the ASS1 BL21 strain is taken out for use and is recovered and cultured to OD₆₆₀Adding IPTG with final concentration of 1mM to 0.4-0.6, inducing for 4h, centrifuging, collecting bacterial liquid, and storing at-80 deg.C;

(2) and (3) protein extraction: thawing a bacterial solution stored at the temperature of minus 80 ℃, adding a lysine buffer flushing solution to resuspend bacteria, adding lysozyme, placing on ice, ultrasonically breaking the wall, centrifuging, taking a supernatant, and standing on ice for later use;

(3) and (3) protein purification: adding Ni-NTA filler into the supernatant, incubating, loading the mixture into a column, eluting, and collecting the eluate;

(4) desalting and preserving of proteins: ultrafiltering, centrifuging, desalting and diluting the eluate;

(5) subpackaging and storing: measuring the concentration of the diluted protein, subpackaging, quick freezing with liquid nitrogen, and storing at-80 deg.C.

9. Use of a spinosyn derivative and pharmaceutically acceptable salts thereof for the use according to any of claims 1-5 in the manufacture of a medicament for the treatment of tumors associated with argininosuccinate synthetase.

10. Use of a spinosyn derivative and pharmaceutically acceptable salts thereof for the use of any of claims 1-5 in the manufacture of a medicament for the treatment of type I citrullinemia associated with argininosuccinic acid synthetase.