CN105153042B - A kind of compound and its purposes in the medicine for preparing treatment cocaine habituation - Google Patents
A kind of compound and its purposes in the medicine for preparing treatment cocaine habituation Download PDFInfo
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- CN105153042B CN105153042B CN201510458757.XA CN201510458757A CN105153042B CN 105153042 B CN105153042 B CN 105153042B CN 201510458757 A CN201510458757 A CN 201510458757A CN 105153042 B CN105153042 B CN 105153042B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/50—Three nitrogen atoms
Abstract
The invention discloses a kind of compound, and the invention also discloses the purposes of the compound and its medicine of preparation.The compounds of this invention specific can suppress PRMT1, PRMT1 inhibitor can be used as, reduce H4R3me2a and cocaine habituation related gene Cdk5 and CaMK II expression, cocaine award behavior can effectively be weakened, it can prepare good as drug therapy cocaine habituation, potential applicability in clinical practice.
Description
Technical field
A kind of purposes the present invention relates to new compound and its in the medicine for preparing treatment cocaine habituation.
Background technology
Cocaine（Cocaine）Also known as cocaine, the entitled benzoyl-methyl-ecgonine of chemistry（methyl
benzoylecgonine）, general white lenticular is odorless, bitter and it is numb, it is used for local anaesthesia and treatment heavy breathing earliest
Asthma, be most strong natural central stimulant, cause to abuse because of the excitation of its Central nervous system, from 1985 into
For one of worldwide main drugs.
Cocaine habituation, it is a kind of chronic recurrent brain diseases, belongs to pharmacological dependence（drug dependence）Class disease
Disease, cocaine habituation can cause the Changes of Plasticity of brain structure and function, and related brain areas includes nucleus accumbens septi, corpus straitum, forehead
Cortex, hippocampus and ventral tegmental area, health are also disorderly by many harm, including mental deterioration, personality defect, intelligence function
Disorderly, concurrent corresponding infection complication and drug addict seek and use drugs and induce various delinquent work at all adventures
It is dynamic.
Even if the main feature of cocaine habituation shows as patient after the serious consequence of medication is known, sex cords is still forced
Take and use with meet desire, to medicine seek and ask for it is out of hand, things is lost interest, Drug addiction it is very deep
Carve, after the withdrawal and treatment several years, the contact stimulation relevant with habituation（Such as malicious friend, the environment relevant with medication in the past
Deng）It may all induce and relapse.
In view of cocaine habituation harm is very big, suitable therapy target and medicine are found, treatment cocaine habituation is compeled in eyebrow
Eyelash.
The content of the invention
In order to solve the above problems, the invention provides a kind of compound, can treat cocaine habituation.
PRMT1：Protein arginine methylated transferase 1.
PRMT1 inhibitor：Suppress the enzyme activity of protein arginine methylated transferase 1 or the material of expression.
The compounds of this invention, its structural formula are as follows：
Present invention also offers purposes of the aforesaid compound in PRMT1 inhibitor is prepared.
Present invention also offers purposes of the aforesaid compound in the medicine of cocaine habituation is prepared.
Preferably, the medicine is the medicine for the expression for reducing H4R3me2a.It is further preferred that the medicine is
Reduce the medicine of gene C dk5 and CaMK II expression.
Present invention also offers a kind of medicine for treating dependence producing drug habituation, and it is that aforesaid compound is active component, is added
The preparation that upper pharmaceutically acceptable auxiliary material is prepared.
Preferably, the preparation is ejection preparation.
Present invention also offers purposes of the foregoing pharmaceutical in PRMT1 inhibitor is prepared.
Present invention also offers purposes of the foregoing pharmaceutical in the medicine of cocaine habituation is prepared.
Preferably, the medicine is the medicine for the expression for reducing H4R3me2a.It is further preferred that the medicine is
Reduce the medicine of gene C dk5 and CaMK II expression.
The compounds of this invention specific can suppress PRMT1, can be used as PRMT1 inhibitor, reduce H4R3me2a and cocker
Because of habituation related gene Cdk5 and CaMK II expression, cocaine award behavior can be effectively weakened, can prepare turns into
Drug therapy cocaine habituation, potential applicability in clinical practice are good.
The embodiment of form by the following examples, the above of the present invention is made further specifically
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following embodiment.It is all to be wanted based on right of the present invention
The technology that the content for asking secretary to carry is realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 mouse conditioned place preference casees.
Fig. 2 spontaneous activity inspection boxs.
Injection MTA suppresses the conditioned place preference behavior of cocaine induction in Fig. 3 nucleus accumbens septis.(A) Conditioned place is inclined
Like model manipulation schematic diagram, MTA (500 μM, 1 μ l/ are only) shifts to an earlier date in 30min nucleus accumbens septis and injected；(B) MTA is obviously reduced cocaine
The award behavior of induction, One-way ANOVA, * * p<0.01, * * * p<0.001, n.s. represents no significant difference.
Vehicle+saline groups n=9, MTA+saline group n=8, vehicle+cocaine group n=8, MTA+cocaine group n=
11.(C) MTA promotes the regression of cocaine habituation mouse award behavior, Two-way ANOVA, * * * p<0.001, Vehicle+
Saline groups n=9, MTA+saline group n=8, vehicle+cocaine group n=9, MTA+cocaine group n=8.
Injection AMI-1 suppresses the conditioned place preference of cocaine induction in Fig. 4 nucleus accumbens septis.(A) conditioned place preference
Model manipulation schematic diagram, AMI-1 (1mM, 1 μ l/ are only) shift to an earlier date in 30min nucleus accumbens septis and injected；(B) AMI-1 is obviously reduced cocaine
The award behavior of induction, One-way ANOVA, * * p<0.01, * * * p<0.001, n.s. represents no significant difference.
Vehicle+saline groups n=13, AMI-1+saline group n=9, vehicle+cocaine group n=12, AMI-1+cocaine
Group n=12.
Injection AMI-1 suppresses the H4R3me2a expression of cocaine induction in Fig. 5 nucleus accumbens septis.(A) AMI-1 suppresses cocker predisposition
The H4R3me2a up-regulated expressions led, One-way ANOVA, * p<0.05, Vehicle+Saline group n=4, AMI-1+Saline
N=4, Vehicle+Cocaine n=4, AMI-1+Cocaine n=4；(B) AMI-1 does not influence PRMT1 expression, One-
Way ANOVA, * p<0.05, n.s represents no significant difference, Vehicle+Saline n=4, AMI-1+Saline n=
4, Vehicle+Cocaine n=4, AMI-1+Cocaine n=4.
Fig. 6 SKLB-639 synthetic lines
Fig. 7 SKLB-639 suppress PRMT family proteins activity in vitro.A-F be SKLB-639 to PRMT1, PRMT3, PRMT4,
PRMT5, PRMT6 and PRMT8 dose-response figure, SKLB-639 detectable concentration scope is 0.005,0.046,0.137,
0.412nd, 1.235,3.704,11.111,33.333 and 100.000 μM.
Fig. 8 SKLB-639 and hPRMT1 docking schemes.(A) SKLB-639 interacts with PRMT1 substrate binding pockets, with reference to
The no color mark in region：SAM binding sites (yellow, amino acid residue His53, Arg62, Glu86, Glu 108,
Glu 137) and avtive spot (magenta, amino acid residue are Glu152and Glu161)；(B) SKLB-639 is in PRMT1 eggs
Substrate binding site on white, including Glu161, Met154, Tyr47, His301, Arg361and Tyr156.
Fig. 9 SKLB-639 suppress the mechanism of action (MOA) of PRMT1 enzymatic activitys.(A) SKLB-639 IC50With polypeptide bottom
Thing concentration increases and linearly raised, and illustrates SKLB-639 and peptide substrate competitive binding to PRMT1；(B) SKLB-639
IC50Increase not significant change with SAM concentration, illustrate SKLB-639 not with SAM competitive bindings PRMT1.
Injection SKLB-639 suppresses the conditioned place preference behavior of cocaine induction in Figure 10 nucleus accumbens septis.(A) conditionity
Place Preference model manipulation schematic diagram, SKLB-639 (1mM, 1 μ l/ are only) shift to an earlier date in 30min nucleus accumbens septis and injected；(B)SKLB-639
It is obviously reduced the award behavior of cocaine induction, One-way ANOVA, * p<0.05, * * * p<0.001, n.s. represents not unite
Meter learns difference.Vehicle+saline groups n=13, SKLB-639+saline group n=10, vehicle+cocaine group n=11,
SKLB-639+cocaine groups n=13.
The regulation and control nucleus accumbens septi PRMT1 histone substrates modifications of Figure 11 cocaines.(A) cocaine increases H4R3me2a modification,
Actin is internal reference albumen, Student ' s t tests, * p<0.05, n=4；(B) cocaine does not influence repairing for H2AR3me2a
Decorations, Actin are internal reference albumen, n=4.
Specificity suppresses H4R3me2a modification in Figure 12 SKLB-639 bodies.(A) SKLB-639 reduces H4R3me2a table
Reach, * p<0.05,**p<0.01, n.s. represents no significant difference；(B-D) SKLB-639 is not influenceed in nucleus accumbens septi
H3R2me2a, H3R17me2a and the arginic modification of histone symmetry di-methylation.Vehicle+Saline n=4,
Vehicle+Cocaine n=4, SKLB-639+Cocaine n=4, SKLB-639+Saline n=4.
Figure 13 H4R3me2a and acH3K9/K14 regulate and control Cdk5 genetic transcriptions.(A) in cocaine repeat administration model,
H4R3me2a and acH3K9/K14 is combined in Cdk5 gene promoters site to be increased, Students ' t-test, * p<0.05, N=
3/ group, 4 animal nucleus accumbens septi samples are incorporated to a sample (being considered as N=1)；(B) cocaine repeat administration up-regulation nucleus accumbens septi
Cdk5mRNA expression, Students ' t-test, * p<0.05, n=5/ group.
Figure 14 H4R3me2a and acH3K9/K14 regulate and control CaMK II genetic transcriptions.(A) in cocaine repeat administration model
In, H4R3me2a and acH3K9/K14 combine increase, Students ' t-test, * p in CaMK II gene promoters site<
0.05, N=3/ group, 4 animal nucleus accumbens septi samples are incorporated to a sample (being considered as N=1)；(B) cocaine repeat administration up-regulation volt
Every core CaMK II mRNA expression, Students ' t-test, * p<0.05, n=5/ group.
Figure 15 cocaine repeat administrations increase H4R3me2a expression.Cocaine repeat administration increase nucleus accumbens septi H4R3me2a
Expression, the expression status for increasing H4R3me2a at least maintained after Cocaine withdrawal the 7th day.The nucleus accumbens septi pattern detection time point
Not Wei Cocaine withdrawal 1 day (D1), 7 days (D7), 14 days (D14), 28 days (D28) and 42 days (D42), Actin is as internal reference egg
In vain, * p<0.05 shows there is significant difference compared with physiological saline group, n=3/ groups.
Figure 16 cocaines repeat administration reduces nucleus accumbens septi H3K9me2 expression.The expression for reducing H3K9me2 is guarded against in cocaine
Recover normal level within disconnected 7th day.The nucleus accumbens septi pattern detection time be respectively Cocaine withdrawal 1 day (D1), 7 days (D7), 14 days
(D14), 28 days (D28) and 42 days (D42), Actin is as albumen internal reference, * p<0.05 shows have compared with physiological saline group
Significant difference, n=3/ groups.
Figure 17 cocaines repeat administration reduces nucleus accumbens septi H3K36me3 expression.H3K36me3 expression is reduced to guard against in cocaine
Recover normal level within disconnected 7th day.The nucleus accumbens septi pattern detection time be respectively Cocaine withdrawal 1 day (D1), 7 days (D7), 14 days
(D14), 28 days (D28) and 42 days (D42), Actin is as albumen internal reference, * p<0.05 shows have compared with physiological saline group
Significant difference, n=3/ groups.
Embodiment
The compounds of this invention SKLB-639 of embodiment 1 preparation method
1.6g4- aminobenzene carbonamidines dihydrochloride, 1.8mL triethylamines, 150mL ethanol is added in 250mL round-bottomed flasks to stir
Mix uniformly, then add 0.5g4, the chloro- 5- nitro-pyrimidines of 6- bis-, reaction solution, which is placed at 50 DEG C, to react 3 hours, there are a large amount of yellow to consolidate
Body is separated out, and then stops reaction, and cooled and filtered extracting yellow solid is washed three times with ethanol, then obtained most with recrystallizing methanol
Finished product, yellow, yield 60%.
1H NMR(400MHz,DMSO)：δ10.84(s,2H),9.39(s,4H),9.16(s,4H),8.30(s,1H),
7.89(s,8H)
13C NMR(101MHz,DMSO)：δ167.11,164.23(2C),163.07(2C),141.23(2C),126.76
(4C),118.07(2C),111.16(2C),111.34(4C)
ESI-MS：392.1[M+H]+
The compounds of this invention SKLB-639 structural formula：
Hereinafter beneficial effects of the present invention are verified with the mode of experimental example：
The compounds of this invention SKLB-639 of experimental example 1 treats cocaine habituation
1 experiment reagent
The reagent that this part uses such as table 1-1.
Table 1-1 experiment reagents
2 key instruments
The laboratory apparatus that this part uses such as table 1-2.
Table 1-2 laboratory apparatus
3 experimental methods
3.1 experimental animal
Male SPF levels health sexal maturity (8-12 week old) C57BL/6J mouse are by the limited duty of Shanghai City Si Laike experimental animals
Ren companies provide, and body weight 20-22g, do not mate.Rearing conditions：National Chengdu new drug preclinical safety evaluation center is common
Animal House, 20-25 DEG C of temperature, relative humidity 55-65%, in whole experiment process, animal is own to ingest and drinks water, feeding environment
Meet national standard GB14925-2001.All zoopery operations of this problem meet AAALAC requirements, are normally raised before experiment
Experimental animal is for a period of time to be familiar with and adapt to environment.
3.2 intracerebral injection PRMT1 inhibitor
By the mouse of unilateral nucleus accumbens septi pipe laying, using 33 type micro-injection pins and micro-injection conduit, given according to table 1-3
Pharmaceutical quantities give different PRMT1 inhibitor --- MTA, AMI-1 and SKLB-639.MTA, AMI-1 and SKLB-639 DMSO
100X storing solution is formulated as, using preceding with normal saline dilution to usable concentration.
Table 1-3 PRMT1 inhibitor injection dosages
3.3 nucleus accumbens septi brain area pipe layings
Brain area positioning pipe laying is the direct approach and common technology of brain area site-specific delivery of drugs.10% hydration chlorine is used in this research
Aldehyde (0.4g/kg body weight) anesthetized mice, overhead shaving, postanesthetic mouse is fixed on stereotaxic apparatus, after fixed
Head is adjusted to horizontality, medicinal alcohol sterilization skin, surgical exposure skull, skull surface is removed with cotton balls and eye scissors
Meninx, find bregma position, marking pen mark；Stereotaxic instrument is adjusted, disposes injection catheter (Shenzhen Rui Wode biotechnologies
Co., Ltd, #62003, OD 0.48mm X ID 0.34mm), returned to zero in bregma；Using bregma position as origin, reference location
Coordinate (nucleus accumbens septi：AP+1.6；ML+1.5) injection needle is moved to the surface of injection site, makes injection needle and skull rigid connection
Touch, return to zero Y-coordinate, slowly moves downward injection needle to 3.4mm depths, unclamps coordinate arm and simultaneously remove, dentistry powder and dilution are adjusted
To starchiness fixed sleeving bottom, it is connected with skull and reaches the purpose of A/C.Insert conduit cap (Shenzhen Rui Wode biologies section
Skill Co., Ltd, #62102, OD 0.30mm), prevent catheter blockage, sewing-end top wound.After operation terminates, mouse is protected
Temperature is then placed in clean rearging cage to reviving, single single cage raising.Since Post operation second day, daily rear leg muscle
Benzylpenicillin sodium salt solution (160000U/ml, 0.1ml/ mouse) is injected, and loosens conduit cap every other day, it is ensured that clearness of catheter, mouse
The convalescence after Miles operation be 7 days.Animal by this operation is used to studying nucleus accumbens septi injection PRMT1 inhibitor (including MTA, AMI-1
And SKLB-639) regulation and control Cocaine-induced Conditioned Place Preference behavioral value.
3.4 nucleus accumbens septi locating injection slow virus
10% chloraldurate (0.4g/kg body weight) anesthetized mice, overhead shaving, is fixed on brain by postanesthetic mouse and stands
On body position indicator, regulation head to horizontality, medicinal alcohol sterilization skin, surgical exposure skull, with cotton balls and eye scissors
The meninx of skull surface is removed, finds bregma position, marking pen mark；Stereotaxic instrument is adjusted, according to the elements of a fix (AP+
1.6；ML±1.5；DV-3.4), it is sick slowly to inject 0.5 μ l in 0.1 μ l/min speed bilateral nucleus accumbens septis using 33 type injection needles
Poison, after injection, slowly remove injection needle, sewing-end top skin.After operation terminates, insulation mouse to revival, Ran Houfang
Enter in clean rearging cage, single single cage raising.Since Post operation second day, daily rear leg intramuscular injection Benzylpenicillin sodium salt solution
(160000U/ml, 0.1ml/ mouse), continuous injection 3 days, the convalescence after Miles operation of mouse is 7 days.By the animal of this operation
Suppress PRMT1 expression slow virus regulation and control Cocaine-induced Conditioned Place Preference behaviouristics detections and spontaneous for studying nucleus accumbens septi injection
Crawler behavior detects.
3.5 cocaine inducing mouse Conditioned place preference models (CPP)
CPP mouse cocaine habituation models are modified slightly after establishing bibliography methodology[32], CPP operations are such as Fig. 1 institutes
Show.Black, the white two big case and a middle ash bin that CPP chambers are made by lucite are formed, and a chest has black
The extra coarse ground of four walls and the round-meshed rough earth of tool, another white four wall and stripe.Mouse is free in experimental box
Activity, adapt to three days.CPP experiments are divided into 3 stages, and in experimentation, cocaine and physiological saline are injected according to table 1-4.
First stage (day1)：The natural preference of mouse is tested, under general condition, mouse shows natural preference, institute to black box
Using the training stage below using white box as with medicine-chest.Second stage (day2-day7)：For the training stage, during this period, dividing plate is used
Passage between fully sheathed case, the 2nd, 4,6 day injection cocaine, and mouse is put into white box immediately, the 3rd, 5,7 day injection physiology salt
Water, and mouse is put into black box immediately, each mouse residence time in case is 15min；Phase III (day8)：To survey
In the examination stage, in this stage, the dividing plate between case is removed, make mouse freely movable in case, and record 15min mouse and exist respectively
Residence time in black, white box.After mouse test terminates, in fast anatomical in 30min, take out nucleus accumbens septi and be used for subsequent detection.
As a result during being tested with conditioned place preference, the condition induction casing residence time subtracts the stop of another casing
Time difference is relatively expressed as preference after induction compared with the natural preference time.Experiment knot is analyzed using SPSS statistical softwares
Fruit, difference are represented with mean ± standard deviation, and the difference of cocaine administration group and saline control group is compared with t check analysis methods
It is different, p<0.05 is to have significant difference.
Points for attention：During each animal training, it should handle with care, not cause significant impact to the mood of animal；Often
Secondary training terminates wipe casing, to eliminate the smell of each animal.Intensity of illumination, daily training time, instruction
Practice duration, noise should pay attention to controlling.
Table 1-4 cocaine administrations approach, dosage, administered volume summary sheet
The regression model of 3.6 cocaines award behavior is established
After Cocaine-induced Conditioned Place Preference model is established, physiological saline group animal is randomly divided into physiological saline+solvent
Control group, physiological saline+MTA groups；The cocaine group animal for showing preference is randomly divided into cocaine+solvent control group, can
Cacaine+MTA groups, all animals inject corresponding solvent control or MTA, are put into immediately after immediately after CPP tests are completed
Rearging cage, preference behavior of the mouse to CPP casees is determined again after 24h, and record 15min mouse and stopped respectively in black, white box
Time.
3.7 cocaine spontaneous activity models are established
Before experiment, mouse adapts to detecting instrument (Fig. 2) 3 days, daily 5min.Experiment start after, the 1st day record mouse from
Send out activity value, based on be worth；Later ask to join one daily the time intraperitoneal injection 20mg/kg cocaines, continuous 6 days, 1 time a day.
It is immediately placed in after drug injection in spontaneous activity box, records the behavioral activity (such as operating range) of mouse in 30min.
3.8 chemoluminescence methods detect IC of the micromolecular compound to PRMTs enzymes50
Chemical luminescence reagent kit (BPS Bioscience, PRMT3 (52005L), PRMT4 (52041L), PRMT5
(52002L), PRMT6 (52046), and PRMT8 (52058)) it is used to determine micromolecular compound to corresponding apparent modification enzyme
IC50.Specially：According to chemical luminescence reagent kit operational manual, histone H 4 is covered with advance with 150 μ l TBST aquations first
Reacting hole 15min, then according to measure hole number, reaction substrate mixed liquor (such as table 1-5) is prepared, in last corresponding measure hole
Test compound and reaction enzymes are separately added into, reaction starts.During first screening compounds, the concentration using compound is 10 μ
M, then according to results of preliminary screening, determine the IC for specifying several compounds50When, the concentration range of compound is 0.005,
0.046th, 0.137,0.412,1.235,3.704,11.111,33.333 and 100.000 μM.For this serial enzyme reaction,
PRMT1 enzyme reaction concentration is reacted for 2.5ng/, and PRMT3 enzyme reactions concentration is reacted for 25ng/, and PRMT4 enzyme reaction concentration is
200ng/ is reacted, and PRMT5 enzyme reactions concentration is reacted for 100ng/, and PRMT6 enzyme reactions concentration is reacted for 100ng/, and PRMT8 enzymes are anti-
Concentration is answered to be reacted for 50ng/.Placing response hole is after room temperature 60min, with the 200 μ l TBST board-washings 3 times of Fresh, each
Reacting hole adds 100 μ l confining liquids, vibration closing 10min, topples over confining liquid, adds 100 μ l primary antibodies, be incubated at room temperature 60min, so
200 μ l TBST board-washings 3 times afterwards；100 μ l secondary antibodies are added, are incubated at room temperature 30min, 200 μ l TBST board-washings 3 times, finally according to 1:1
Ratio mix HRP chemical luminous substrates A and B on ice, after each reacting hole adds 100 μ l light-emitting admixtures, immediately in detection
(plate reading machines of BioTek SynergyTM 2) is detected on instrument.Finally, the data obtained to detection calculate analysis according to the following formula.
Testing result processing is carried out according to formula *：%activity=(C-C0)/(CPositive control-C0)X 100*
Compound percent inhibition：%inhibition=100-%activity#
Wherein C is simple chemical luminous value, CPositive controlFor positive control values of chemiluminescence, C0For blank control chemiluminescence
Value.
Table 1-5 micromolecular compounds IC50Detect loading
3.9 protein extractions and western blot (Western Blot)
3.9.1 the extraction of total protein is with quantifying
The tissue frozen is taken, RIPA lysates are added according to tissue size, for nucleus accumbens septi tissue, add 100 μ l or so,
It is placed in and cracks 5min on ice, then ultrasound 10 time, 2 seconds/time (being placed on ice), it is therefore an objective to which disrupting tissue cell, fully dissolving are released
Albumen is put, then 4 DEG C, 13,000g centrifugation 15min, supernatant is taken out, puts on ice.Protein quantification is main referring especially to BCA methods
The step is wanted to be：First, BSA to 1,0.5,0.4,0.3,0.2,0.1,0.05,0mg/ml concentration gradient are diluted；Then by extraction
Protein liquid is according to nucleus accumbens septi (1:10 dilutions), corpus straitum (1:50 dilutions), hippocampus (1:50 dilutions) and prefrontal cortex (1:10 is dilute
Release) dilution, BSA and protein sample are separately added into 10 μ l in respective aperture.Before measure, it is bright that 200 μ l coomassies are added in each hole
Blue G250,595nm determine absorbance, and the existing correlation of BSA standard curves is R2>0.99 is qualified quantitative criterion, Ran Houji
Calculate corresponding protein concentration；Each sample adds 5X albumen sample-loading buffer (containing beta -mercaptoethanol), makes the ultimate density be
1X.Sample 5min is boiled, dispenses, is stored in -20 DEG C.
3.9.2 polyacrylamide gel electrophoresis
According to table 1-6, table 1-7 polyacrylamides prepare the separation gel and 5% concentration glue that glue prepares 10% and 15%.Will
The glue prepared is put into electrophoresis tank, point sample.According to abundance of each albumen in nucleus accumbens septi, sample concentration, each sample are adjusted
It is about 18 μ l that this groove, which adds albumen sample volume,.When then using at 80V voltage compression albumen samples to separation gel boundary, by electricity
Pressure increases to 120V, until destination protein is kept completely separate.When carrying out transferring film program, the glue for being loaded with destination protein is put into and carried
In the transferring film folder of " sandwich ", and the pvdf membrane by methanol activation is covered on glue, transferring film voltage is set as 100V, during transferring film
Between depending on albumen size.After the completion of transferring film, 5% skim milk prepared with TBST is closed, and off-period is 1 hour.
By the dilution proportion of primary antibody as required, sealed with hybrid belt, 4 DEG C overnight, are washed film three times in second day, each 10min with TBST.
According to primary antibody source property, according to 1:The 5000 corresponding secondary antibodies of dilution, room temperature 2h, it is secondary with TBST to wash film after terminating, every time
10min, then wash film once with TBS, same 10min.During exposure, according to 1:1 ratio prepares luminous A liquid and luminous B liquid,
The PVDF bands for being loaded with destination protein are placed in luminous mixed liquor in darkroom and react 1min, pvdf membrane is taken out, inhaled with filter paper
Unnecessary luminescent solution is done, according to face-up principle, pvdf membrane is put into magazine, film front also film contact, closes magazine,
The closing film time depends on protein abundance, develops a film.
Data processing：Western blotting film is read to the gray value of each band with the systems of Image-Pro Plus 6.0, used
Beta-actin band gray values are standardized as internal reference to be compared.
Table 1-6 Western blot separation gels prepare configuration
Table 1-7 Western blot concentration glue prepares configuration
3.10 real-time fluorescence quantitative PCR
3.10.1 RNA is extracted
For brain tissue, brain tissue is put into existing 1ml Trizol 1.5ml EP pipes, carrys out blowback using 1ml pipette tips
Beat nearly 100 times, histocyte is cracked as far as possible.For cell culture, nutrient solution is sucked, adds 1ml Trizol, room temperature places 5
Cell is blown and beaten repeatedly after minute, cell is cracked as far as possible, and cell lysate is moved into a 1.5ml EP pipe.Then tissue is split
Solve liquid and cell pyrolysis liquid adds 200 μ l chloroforms, vibrate 15 seconds, room temperature is placed 2~3 minutes；4 DEG C, 12000g, 15min is centrifuged,
After centrifugation, liquid is divided into three layers, and RNA is present in the aqueous phase of the superiors, and the volume of aqueous layer is about when being homogenized
The 60% of Trizol volumes.Aqueous layer is transferred in a new EP pipe (1.5ml), is careful not to pollute intermediate layer, before
The isopropanol that 0.5ml is added in 1ml Trizol used precipitates RNA, and 10min or 4 DEG C is incubated at room temperature after mixing and is incubated 20
~30min, 4 DEG C, 12000g centrifugation 10min, RNA precipitate agglutination is attached on ttom of pipe.Supernatant is outwelled, wink is from exhaustion residual.
1ml Trizol at least add the ethanol of 1ml 75% washing RNA, 4 DEG C, 10000g, centrifuge 5min.Supernatant is outwelled, wink is from exhaustion
Residual.And the drying at room temperature 10min that uncaps, it is careful not to overdrying.RNA, the RNA samples of purifying are dissolved using 20 μ l DEPC water
Originally -80 DEG C are stored in.
3.10.2 agarose gel electrophoresis
Electrophoresis tank used, comb, glue plank, conical flask, graduated cylinder (100ml, 1000ml) are ready to and cleaned
(running water, deionized water and milic water), then load onto millic water and TAE is diluted for after.Appropriate agarose is weighed in cone
In shape bottle, 1X TAE solution is added by required concentration and volume, can configure 25-30ml (1% gum concentration, i.e. 0.25-0.3mg),
Melt in micro-wave oven.When slightly cold, 5 μ l Gold View dyestuffs are added, jog is even, avoids producing bubble.It is cooled to about 60
DEG C when fall glue (thickness is within 0.5cm), when solidifying completely, extract comb, loading electrophoresis, electrophoretic buffer should not have glue
Plate.Sample-adding, sample loading buffer are no less than 1X.Electrophoretic voltage (sample is (red) mobile from negative pole (black) to positive pole) 200V 7min,
After sample runs out of loading wells about 1-2cm, Ago-Gel is imaged with IMAGE LAB.
3.10.3 Realtime PCR react
The Reverse Transcriptase kit synthesis cDNA (Bio-rad) of commodity in use, the cDNA of synthesis are stored in -80 DEG C.Then
Realtime PCR reactions are carried out, the gene mRNA primer sequence that this part uses such as table 1-8, all cDNA samples are distinguished
Configure reaction system.System configurations are as follows：2X SYBR green mix 10 μ l, 10 μM of PCR primer F 1 μ l, 10 μM of PCR draw
The μ l of 1 μ l, cDNA samples of thing R 1, add water to the μ l of cumulative volume 20, flick ttom of pipe and mix solution, carefully cover PCR pipe lid,
The of short duration centrifugations of 5000rpm, ready PCR plate is placed on ice before PCR programs are set.Above-mentioned 96-PCR plates are placed in
The enterprising performing PCR reaction of realtime PCR instruments, reacts and is carried out according to following procedure：95 DEG C of 3min, and 40 PCR cycles (95 DEG C, 15
Second；60 DEG C, 45 seconds (collection fluorescence)).In order to establish the solubility curve of PCR primer, after amplified reaction terminates, by slow from 65 DEG C
It is heated to 95 DEG C.
Table 1-8 gene mRNA primer sequence lists
3.10.4 result is with calculating
The target gene and internal reference of each sample carry out Realtime PCR reactions respectively, and data use 2-ΔΔCtMethod is divided
Analysis.
3.11 data statistics
Significant difference is carried out by the statistical softwares of IBM SPSS Statistics 21.Compare the statistics of two experimental groups
Difference is learned, uses Students ' t to examine；The experimental group for comparing three and the above uses one-way analysis of variance (One-way
ANOVA) Tukey post hoc test are carried out, including CPP behaviouristics data, Western blot data, mRNA results point
Analysis.For the result of spontaneous activity, two-way analysis of variance (two-way is done using the softwares of GraphPad Prism 5
ANOVAs)bonferroni post-tests.All results are with mean value ± SEM (* p in picture<0.05, * * p<
0.01, * * * p<0.001) represent.
4 results
4.1 suppress the award behavior of PRMTs enzymatic activitys reduction cocaine induction
In order to further investigate effects of the PRMT1 in cocaine induction award behavior, this experiment is small using non-specific PRMTs
Molecule inhibitor --- MTA and AMI-1, they can effectively suppress PRMTs enzymatic activitys[48,55,56].Test result indicates that carry
Injection MTA can suppress the award behavior (Fig. 3 A) of cocaine induction, but individually injection MTA in nucleus accumbens septi in preceding 30min nucleus accumbens septis
(Fig. 3 B) is not influenceed on CPP scores.Continue to study the regression for awarding behavior that MTA induces cocaine.By building cocker
Because of CPP habituation mouse, the mouse of habituation is randomly divided into physiological saline group and MTA groups, after cocaine CPP measure, abdominal cavity immediately
After injecting normal saline or MTA, 24h, the CPP scores of mouse are determined again, to evaluate regression of the MTA to cocaine habituation mouse
Behavior.Test result shows that MTA promotes the regression of award behavior caused by cocaine, and independent MTA groups do not have shadow to the behavior
Ring (Fig. 3 C).In order to confirm MTA result, using another " star " PRMT inhibitor --- AMI-1, same use shift to an earlier date
Injection 1mM AMI-1 (Fig. 4 A) administering mode in 30min nucleus accumbens septis.Consistent with MTA results, AMI-1 significantly inhibits cocaine
CPP scores, the interior injection AMI-1 of independent nucleus accumbens septi do not influence (Fig. 4 B) to CPP scores.
PRMT1 can modify H4R3 and obtain H4R3me2a (the 3rd arginine asymmetry di-methylation of histone H 4), this
Experiment detects H4R3me2a expression using Western blot methods.Test result indicates that compared with control group, cocaine group
H4R3me2a expression is increased, and individually injecting AMI-1 groups H4R3me2a expression reduces；Compared with cocaine group, AMI-1 joint cocker
Because reducing H4R3me2a expression (Fig. 5 A).Literature research points out that AMI-1 suppresses PRMT1 enzymatic activitys, but does not have to PRMT1 expression
Have an impact.In order to understand whether AMI-1 influences nucleus accumbens septi PRMT1 expression, its expression is detected using Western blot.
As shown in Figure 5 B, compared with physiological saline group, cocaine increases nucleus accumbens septi PRMT1 expression, individually injects AMI-1 groups PRMT1
Protein expression does not change；Compared with cocaine group, AMI-1 joint cocaine group PRMT1 expression reduces.These results show：
AMI-1 suppresses PRMT1 enzymatic activitys, does not influence nucleus accumbens septi PRMT1 protein expressions；PRMTs is by controlling the way that H4R3me2a is modified
The award behavior of footpath regulation and control cocaine induction.
The 4.2 selective PRMT1 inhibitor of exploitation
4.2.1 Computer-Aided Drug Design screens to obtain SKLB-639
Computer-Aided Drug Design screens to obtain the compound with 5- nitro-pyrimidine -4,6- diamine skeleton symmetrical structures
SKLB-639 (Fig. 6).
SKLB-639 preparation method is as follows:Added in 250mL round-bottomed flasks 1.6g4- aminobenzene carbonamidines dihydrochlorides,
1.8mL triethylamines, 150mL ethanol stir, and then add 0.5g4, the chloro- 5- nitro-pyrimidines of 6- bis-, and reaction solution is placed in 50 DEG C
Lower reaction 3 hours, has a large amount of yellow solids to separate out, and then stops reaction, and cooled and filtered extracting yellow solid washs three with ethanol
It is secondary, then obtain final products, yellow, yield 60% with recrystallizing methanol.
1H NMR(400MHz,DMSO)：δ10.84(s,2H),9.39(s,4H),9.16(s,4H),8.30(s,1H),
7.89(s,8H)
13C NMR(101MHz,DMSO)：δ167.11,164.23(2C),163.07(2C),141.23(2C),126.76
(4C),118.07(2C),111.16(2C),111.34(4C)
ESI-MS：392.1[M+H]+
In order to contrast SKLB-639 and AMI-1 under the conditions of same detection to PRMT1 external 503nhibiting concentration (IC50),
Two compounds are sent to U.S. BPS Bioscience (San Diego, CA, United States) company and carry out active survey
Examination.Test result shows, under the conditions of same detection, SKLB-639 IC50For 2.4 μM, activity is apparently higher than " star suppresses
Agent " AMI-1 (IC50=20 μM) about 8 times (1822A), show that SKLB-639 has high inhibitory activity to PRMT1.
4.2.2 SKLB-639 specificity suppresses PRMT1
In order to study SKLB-639 to PRMT1 specific inhibitory effects, SKLB-639 pairs is detected using in-vitro method
PRMT3, PRMT4, PRMT 5, PRMT 6, PRMT8 IC50.Because PRMT2 and PRMT7 do not have enzymatic activity or with extremely low
Enzymatic activity, do not detect herein.Testing result shows that SKLB-639 is 36.4 μM to PRMT3 503nhibiting concentration, with PRMT1 ratios
Relatively have>15 times of selectivity (Fig. 7 B)；SKLB-639 is 73.9 μM (Fig. 7 C) to PRMT4 503nhibiting concentration, compared with PRMT1
Have>30 times of selectivity；SKLB-639 is all higher than 100 μM to PRMT5, PRMT6 and PRMT8 503nhibiting concentration, shows do not have
Inhibitory action (Fig. 7 D-F).Data above shows：SKLB-639 efficients and high selectivity suppress PRMT1.
4.2.3 SKLB-639 suppresses PRMT1 mechanism of action with docking research
Molecular docking is analyzed from the measurements of the chest, waist and hips conformation of protein and drug molecule, and whether investigate can tie between them
Close, and predict the binding pattern with reference to compound, be an analysis means useful in CADD.Using computer to SKLB-639
Carry out docking research, the results showed that, SKLB-639 is combined (Fig. 8) with hPRMT1 substrate binding pocket.PRMT1 binding pockets
By side chain Tyr47, Ile52, Met56, Trp302, His301 and framework residue Glu152-Tyr156 be collectively forming one it is narrow
Passage, with PRMT1Glu161 conserved active sites (and Double-E structures) electrostatic occurs for a SKLB-639 amidine group
Reacted with hydrogen bond, occupy binding pocket.In addition, SKLB-639 also with PRMT1 residues Met154, Tyr47and His301
Hydrogen bond action is formed, wherein Met154 is the active residue that another PRMT guards with arginine substrate binding site.Nitro base
The presence of group forms hydrogen bond with Tyr156.With other asymmetry and there is 5-nitropyrimidine-4,6-diamine bones
Frame compound compares, and presence and PRMT1 the albumin As rg361 and Tyr163 of another amidine group of SKLB-639 form hydrogen bond, this
The formation of hydrogen bond is probably the reason for SKLB-639 selective depression PRMT1.
Entrust U.S. BPS Bioscience (San Diego, CA, United States) biotech firm detection SKLB-
639 and PRMT1 mechanism of action (MOA, mechanism of action) and between the two possible binding pattern.As a result
Show, with linearly increasing, ICs of the SKLB-639 to PRMT1 of histone H 4 concentration of substrate50Linearly increase (Fig. 9 A)；With
The change of confactor SAM concentration, ICs of the SKLB-639 to PRMT150Do not change (Fig. 9 B).These results show：SKLB-
639 suppress PRMT1 activity by substrate competition, rather than the mechanism of confactor competition；Illustrate SKLB-639 and PRMT1 simultaneously
Binding pocket combines, and confirms the protein bound guesses of SKLB-639 and PRMT1.
4.3 SKLB-639 suppress the award behavior of cocaine induction
Acted on to study inside SKLB-639, using Cocaine-induced Conditioned Place Preference model evaluation its in cocaine
Effect in habituation.It is identical with MTA and AMI-1 administering mode, (schemed using SKLB-639 is injected in 30min nucleus accumbens septis in advance
10A).Behaviouristics result shows that SKLB-639 reduces the CPP scores of cocaine induction, but individually injection SKLB- in nucleus accumbens septi
639 do not influence CPP scores (Figure 10 B).
In order to understand SKLB-639, whether specificity suppresses internal PRMT1, and this experiment utilizes Western blot detection volts
Every core brain area H4R3me2a (PRMT1 modifies substrate), H2AR3me2a (PRMT1 modifies substrate), (PRMT4 is modified H3R17me2a
Substrate), H3R2me2a (PRMT6 modify substrate) and histone symmetry di-methylation arginine (II types PRMTs modifications substrate)
Expression.Testing result shows that cocaine adds nucleus accumbens septi H4R3me2a modification (Figure 11 A), but does not influence H2AR3me2a
Modification (Figure 11 B)；Compared with cocaine group, the H4R3me2a that SKLB-639 reduces cocaine induction increases degree, and
Independent SKLB-639 groups lower H4R3me2a expression (Figure 12 A).SKLB-639 to other histone substrates H3R17me2a,
H3R2me2a and the modification of symmetry di-methylation are all without influence (Figure 12 B-D).Result above shows：Injected in nucleus accumbens septi
SKLB-639 specificity suppresses PRMT1 substrates H4R3me2a modification, and then regulates and controls the award behavior of cocaine induction.
5 conclusions
Experimental result illustrates that the compounds of this invention SKLB-639 can specifically suppress PRMT1 enzyme activity, and activity is about existing
There are 8 times of inhibitor AMI-1, PRMT1 inhibitor can be made, lower H4R3me2a expression, to suppress cocaine induction
Award behavior, treat cocaine habituation.
Regulating and controlling effects of the H4R3me2a of experimental example 2 to habituation related gene expression
1 experiment reagent
The reagent that this part uses such as table 2-1.
Table 2-1 experiment reagents
2 laboratory apparatus
The instrument that this part uses such as table 2-2.
Table 2-2 laboratory apparatus
3 experimental methods
3.1 chronic intraperitoneal injection cocaines
Dosage regimen is specially：With Section 3.2 of embodiment 1, sample is examined for ChIP, RT-qPCR and Western blot
Survey.
3.2 Cocaine withdrawal
C57 mouse receive 20mg/kg cocaine duplicate injections, after last time is injected, respectively at the 1st day, the 7th day,
14th day, the 28th day and the 42nd day materials nucleus accumbens septi, is detected, as Cocaine withdrawal 1 day (D1), 7 for Western blot
My god (D7), 14 days (D14), 28 days (D28) and 42 days (D42).
3.3 protein extractions and western blot (Western Blot)
3.3.1 the extraction of total protein is with quantifying
The nucleus accumbens septi tissue frozen is taken, 100 μ l RIPA lysates is added, is placed in and cracks 5min on ice, ultrasound 10 times, 2
Second/time (being placed on ice), it is therefore an objective to disrupting tissue cell, fully dissolving release albumen, then 4 DEG C, 13,000g centrifugation 15min,
Supernatant is taken out, is put on ice.Protein quantification mainly comprises the following steps referring especially to BCA methods：First, dilution BSA to 1,0.5,0.4,
0.3,0.2,0.1,0.05,0mg/ml concentration gradient；Then by the protein liquid of extraction according to nucleus accumbens septi (1:10 dilutions), corpus straitum
(1:50 dilutions), hippocampus (1:50 dilutions) and prefrontal cortex (1:10 dilutions) dilution, BSA and protein sample are separately added into 10 μ l
To respective aperture.Before measure, 200 μ l Coomassie brilliant blue G250s, 595nm measure absorbances, BSA standards song are added in each hole
The existing correlation of line is R2>0.99 is qualified quantitative criterion, then calculates corresponding protein concentration；Each sample adds 5X's
Albumen sample-loading buffer (contains beta -mercaptoethanol), and it is 1X to make ultimate density.Boil sample 5min, dispense, be stored in -20 DEG C it is standby
With.
3.3.2 polyacrylamide gel electrophoresis
According to table 2-3, table 2-4 polyacrylamides prepare the separation gel and 5% concentration glue that glue prepares 10% and 15%.Will system
The glue got ready is put into electrophoresis tank, point sample.According to abundance of each albumen in nucleus accumbens septi, sample concentration, each sample are adjusted
It is about 18 μ l that groove, which adds albumen sample volume,.When then using at 80V voltage compression albumen samples to separation gel boundary, by voltage
120V is increased to, until destination protein is kept completely separate.When carrying out transferring film program, the glue for being loaded with destination protein is put into " three
In the transferring film folder of Mingzhi ", and the pvdf membrane by methanol activation is covered on glue, transferring film voltage is set as 100V, transferring film time
Depending on albumen size.After the completion of transferring film, 5% skim milk prepared with TBST is closed, and off-period is 1 hour.Connect
Get off, the dilution proportion of primary antibody as required is sealed with hybrid belt, 4 DEG C overnight, are washed film three times in second day, every time with TBST
10min.According to primary antibody source property, according to 1:The 5000 corresponding secondary antibodies of dilution, room temperature 2h, it is secondary with TBST to wash film after terminating,
Each 10min, then wash film once with TBS, same 10min.During exposure, according to 1:1 ratio prepares luminous A liquid and luminous B
Liquid, the PVDF bands for being loaded with destination protein are placed in luminous mixed liquor in darkroom and react 1min, pvdf membrane are taken out, with filter
Paper blots unnecessary luminescent solution, and according to face-up principle, pvdf membrane is put into magazine, film front also film contact, closes
Magazine, closing film time depend on protein abundance, developed a film.
Data processing：Western blotting film is read to the gray value of each band with the systems of Image-Pro Plus 6.0, used
Internal reference protein band gray value, which is standardized, to be compared.
Table 2-3 Western blot separation gels prepare configuration
Table 2-4 Western blot concentration glue prepares configuration
3.4 real-time fluorescence quantitative PCR
3.3.1 RNA is extracted
Brain tissue is transferred in existing 1ml Trizol 1.5ml EP pipes, nearly 100 are blown and beaten back and forth using 1ml pipette tips
It is secondary, histocyte is cracked as far as possible.For cell culture, nutrient solution is sucked, 1ml Trizol are added, after room temperature is placed 5 minutes
Cell is blown and beaten repeatedly, cell is cracked as far as possible, and cell lysate is moved into a 1.5ml EP pipe.Then to Tissue lysates and
Cell pyrolysis liquid adds 200 μ l chloroforms, vibrates 15 seconds, and room temperature is placed 2~3 minutes；4 DEG C, 12000g, centrifuge 15min, centrifugation
Afterwards, liquid is divided into three layers, and RNA is present in the aqueous phase of the superiors, and the volume of aqueous layer is about Trizol when being homogenized
The 60% of volume.Aqueous layer is transferred in a new EP pipe (1.5ml), is careful not to pollute intermediate layer, it is before used
The isopropanol that 0.5ml is added in 1ml Trizol precipitates RNA, be incubated at room temperature after mixing 10min or 4 DEG C be incubated 20~
30min, 4 DEG C, 12000g centrifugation 10min, RNA precipitate agglutination is attached on ttom of pipe.Supernatant is outwelled, wink is from exhaustion residual.1ml
Trizol at least adds the ethanol of 1ml 75% washing RNA, 4 DEG C, 10000g, centrifuges 5min.Supernatant is outwelled, wink is from exhaustion residual.
And the drying at room temperature 10min that uncaps, it is careful not to overdrying.RNA, the RNA Sample preservations of purifying are dissolved using 20 μ l DEPC water
In -80 DEG C.
3.3.2 agarose gel electrophoresis
Electrophoresis tank used, comb, glue plank, conical flask, graduated cylinder (100ml, 1000ml) are ready to and cleaned
(running water, deionized water and milic water), then load onto millic water and TAE is diluted for after.Appropriate agarose is weighed in cone
In shape bottle, 1X TAE solution is added by required concentration and volume, can configure 25-30ml (1% gum concentration, i.e. 0.25-0.3mg),
Melt in micro-wave oven.When slightly cold, 5 μ l Gold View dyestuffs are added, jog is even, avoids producing bubble.It is cooled to about 60
DEG C when fall glue (thickness is within 0.5cm), when solidifying completely, extract comb, loading electrophoresis, electrophoretic buffer should not have glue
Plate.Sample-adding, sample loading buffer are no less than 1X.Electrophoretic voltage (sample is (red) mobile from negative pole (black) to positive pole) 200V 7min,
After sample runs out of loading wells about 1-2cm, Ago-Gel is imaged with IMAGE LAB.
3.3.3 Realtime PCR react
The Reverse Transcriptase kit synthesis cDNA (Bio-rad) of commodity in use, the cDNA of synthesis are stored in -80 DEG C.Then
Realtime PCR reactions are carried out, the gene mRNA primer sequence that this part uses such as table 2-5, all cDNA samples are distinguished
Configure reaction system.System configurations are as follows：2X SYBR green mix 10 μ l, 10 μM of PCR primer F 1 μ l, 10 μM of PCR draw
The μ l of 1 μ l, cDNA samples of thing R 1, add water to the μ l of cumulative volume 20, flick ttom of pipe and mix solution, carefully cover PCR pipe lid,
The of short duration centrifugations of 5000rpm, ready PCR plate is placed on ice before PCR programs are set.Above-mentioned 96-PCR plates are placed in
The enterprising performing PCR reaction of realtime PCR instruments, reacts and is carried out according to following procedure：95 DEG C of 3min, 40 PCR cycles (95 DEG C,
15 seconds；60 DEG C, 45 seconds (collection fluorescence)).In order to establish the solubility curve of PCR primer, after amplified reaction terminates, by slow from 65 DEG C
Slowly 95 DEG C are heated to.
Table 2-5 gene mRNA primer sequences
3.3.4 result is with calculating
The target gene and internal reference of each sample carry out Realtime PCR reactions respectively, and data use 2-ΔΔCtMethod is divided
Analysis.
3.5 chromatin immunes are co-precipitated (ChIP)
3.5.1 ChIP operating methods
Concrete operation step is：Dissection materials first few minutes prepare the PBS liquid containing 1mM PMSF protease inhibitors, will
Fresh nucleus accumbens septi tissue is put into 500ml PBS (1mM PMSF) solution, and 4 mouse nucleus accumbens septi tissues mix conduct
One tissue samples, tissue is blown and beaten repeatedly with 200 μ l pipette tips, disrupting tissue to fine particle, is then centrifuged for, collect tissue and sink
Form sediment.Using 37% formaldehyde crosslinking 15min, 2M glycine terminating reaction (final concentration of 0.125M), washed and be crosslinked using precooling PBS
Precipitation 3 times, each 5min, centrifugation, precipitation is finally collected, precipitation is placed in SDS protein lysates, then ultrasonication group
To knit, ultrasound condition is adjustment Ultrasonic Cell Disruptor, under conditions of foam is not produced, 15 seconds/time, 50 seconds gaps, totally 10 times, entirely
In ultrasonic procedure, sample is placed on ice bath, the length that the DNA fragmentation obtained by ultrasound is 200bp-500bp, 4 DEG C
13000rpm centrifuges 10min, removes insoluble matter, collects the μ l of supernatant 150, remaining Sample preservation is in -80 DEG C.Take ultrasound broken
The μ l of tissue samples 50 after broken precipitate for H4R3me2a, and 50 μ l precipitate for acH3K9/K14, and 50 μ l are used for negative control, added
Enter in the 450 μ l dilution buffers containing 2.5 μ l protease inhibitors, respectively take 5% to be used for Input and compare.Input controls are placed
Preserved in 4 DEG C of refrigerators, the 20 μ l Protein G magnetic bead by resuspension is separately added into other samples, sample is positioned over 4
It is suspended in DEG C refrigerator overnight, second day, magnetic bead and sample is washed and separated using magnetic frame (Millipore), according to successively suitable
Sequence washs magnetic bead, 500 μ l/ pipe less salt cleaning solutions, 500 μ l/ pipe high salt wash liquid, 500 μ l/ pipe LiCl cleaning solutions and 500 μ l/ pipes
TE cleaning solutions, when washing magnetic bead with TE cleaning solutions, exhaust cleaning solution as far as possible, add and contain in magnetic bead-Protein-DNA mixtures
There are 100 μ l ChIP eluents of 1 μ l Proteinase Ks, be placed in 65 DEG C of water-baths and stay overnight, then 10min in 95 DEG C of water-baths, cool down sample
To room temperature, magnetic bead and supernatant, the EP pipes of professional supernatant to another clean 1.5ml, in each pipe sample are separated using magnetic frame
The 500 μ l actual A of combination is added in this, is fully mixed, in centrifugal filter, 10000g is centrifuged 30 seconds transfer mixed liquor, is abandoned
Fall centrifugate, collect precipitation；Precipitation is washed with washing reagent B, 10000g is centrifuged 30 seconds, discards centrifugate, collects precipitation；Again
10000g is centrifuged 30 seconds, discards centrifuge tube, filter is placed in another clean collecting pipe, 50 are directly added into filter film
μ l elution buffer C, dissolves the DNA of purifying, and 10000g is centrifuged 30 seconds, discards filter, collects liquid, and by fluid preservation
It is used to follow-up PCR in -20 DEG C detect.Realtime-PCR detections, ddH are carried out according to following reaction system2O 8.5 μ l, SYBR-
12.5 μ l, DNA gene primers F of green master mix 1 μ l (table 2-6), the μ l of DNA gene primers R 1, the DNA moulds of purifying
The μ l of plate 2, overall reaction system volume are 25 μ l.After completing sample-adding, flick ttom of pipe and mix solution, carefully cover PCR pipe lid,
The of short duration centrifugations of 5000rpm, ready PCR plate is placed on ice before PCR programs are set.Above-mentioned 96-PCR plates are placed in
The enterprising performing PCR reaction of realtime PCR instruments, reacts and is carried out according to following procedure：95 DEG C of 10min, 50 PCR cycles (95 DEG C,
20 seconds；60 DEG C, 60 seconds (collection fluorescence)).In order to establish the solubility curve of PCR primer, after amplified reaction terminates, by slow from 65 DEG C
Slowly 95 DEG C are heated to.
Table 2-6 gene DNA primer sequences
3.5.2 ChIP results are with calculating
1st, DNA amount is standardized
Δ Ct [normalized ChIP]=(Ct [ChIP]-(Ct [Input]-Log2(Input Dilution
Factor)))
Input Dilution Factor=(fraction of the input chromatin saved)-1
2nd, the Ct values of negative control to be added
Δ Δ Ct [ChIP/NIS]=Δ Ct [normalized ChIP]-Δ Ct [feminine gender]
3rd, the IP Fold Enrichment (enrichment degree) of specific target spot are calculated
Fold Enrichment=2(-ΔΔCt[ChIP/NIS])
4th, S1 and S2 ratio calculation
Δ Δ Ct [S2-S1]=Δ Ct [S2:normalized ChIP]-ΔCt[S1:normalized ChIP]
5th, sample ratio=2(-ΔΔCt[S2-S1])
3.6 data statistics
Significant difference is carried out by the statistical softwares of IBM SPSS Statistics 21.Compare the statistics of two experimental groups
Difference is learned, uses Students ' t to examine；The experimental group for comparing three and the above uses one-way analysis of variance (One-way
ANOVA) Tukey post hoc test are carried out, including Western blot data and mRNA interpretations of result.Own in picture
Result with mean value ± SEM (* p<0.05, * * p<0.01, * * * p<0.001) represent.
4 results
The expression of 4.1 H4R3me2a and acH3K9/K14 regulation and control Cdk5 and CaMK II genes
ChIP experimental techniques are used to detect combination situation of gene transcription regulation in gene promoter area, are adjusted according to gene
Control studies whether it has regulating and controlling effect to the transcriptional control direction of gene.This experiment uses chromatin immune chemical coprecipitation technique
(ChIP) nucleus accumbens septi tissue H4R3me2a and acH3K9/K14 are in Cdk5 and CaMK after detecting cocaine duplicate injection administration 24h
The enrichment degree of II gene promoter areas.As a result show, cocaine increase H4R3me2a and acH3K9/K14 opens in Cdk5 genes
The combination (Figure 13 A) in mover area, cocaine is prompted to strengthen Cdk5 Gene Transcription in vitro.Lied prostrate using RT-qPCR technology for detection
Every the expression of core Cdk5 genes, testing result is shown：Cocaine increase nucleus accumbens septi Cdk5 expression (Figure 13 B).Using same
Detection means and data Study on processing method CaMK II genes, it is found that cocaine also increases H4R3me2a and acH3K9/K14 exists
CaMK II gene promoter areas combine (Figure 14 A), and cocaine repeat administration adds nucleus accumbens septi CaMK II expression (figure
14B).Result above shows：The transcription of H4R3me2a and acH3K9/K14 regulation and control cocaine habituation related genes.
5.2 cocaine repeat administration long periods maintained H4R3me2a modification
In order to evaluate H4R3me2a modifications when long caused by cocaine repeat administration, Cocaine withdrawal mould is built
Type.This experiment uses Western blot technology for detection Cocaine withdrawal nucleus accumbens septis H4R3me2a expression.As a result show,
Compared with physiological saline group, Cocaine withdrawal 1 day and give up 7 days, H4R3me2a expression dramatically increases；Cocaine withdrawal 14 days
When, although H4R3me2a expression increases, do not have significant difference, have started to return to normal level (Figure 15).In order to contrast
Cocaine causes H4R3me2a to increase expression to hold time, and H3K9me2 and H3K36me3 is detected using Western blot methods
Expression.As a result show, compared with physiological saline group, 1 day H3K9me2 and H3K36me3 of Cocaine withdrawal expression reduces, can
Cacaine, which is given up, has recovered normal level (Figure 16 and Figure 17) on the 7th day, prompts cocaine to reduce H3K9me2 and H3K36me3 modification
No more than giving up the 7th day, hence it is evident that the time maintained less than the high expression of H4R3me2a.
Result above shows：Cocaine up-regulation PRMT1 expression, increases H4R3me2a modification, and then regulate and control downstream target base
The transcription of cause, the behavior of final influence cocaine induction are plastic.
5 conclusions
Drug habit is a progressive brain diseases, it is difficult to effect a radical cure, even if after receiving the withdrawal and treatment several years, habituation
Patient still suffers from the high risk relapsed, it is meant that the 26S Proteasome Structure and Function disorder of animal brain is highly stable caused by habituation.Therefore,
One of key challenge of cocaine habituation area research is to find metastable change in the brain, and this is also Many researchers
One of the reason for change depending on gene expression is the important component that habituation is formed.Now, it is this caused by cocaine
Change is considered as " memory of molecule and cell ", because the change of protein molecular can cause the change of gene expression, Jin Erying
Ring the change of neuronal cell morphology and function, this change includes the change of genetic transcription, the change of apparent modification, cynapse can
The change of plasticity, the change of full cell plasticity, the change of neuromorphic, the change of neurotrophic factor balancing.Almost
All these changes all may be relevant with habituation state.
Present invention discover that apparent mark positive influence cocaine habituation related gene Cdk5 and the CaMK II of H4R3me2a table
Reach, the state maintenance time of cocaine increase H4R3me2a expression, which is longer than H3K9me2 and H3K36me3, reduces expression.This result
The interpretation brand-new from one cocaine habituation produces long-term memory.
It is a kind of PRMT1 inhibitor that the compounds of this invention SKLB-639 is demonstrated in experimental example 1, can lower H4R3me2a
Expression, and this experimental example demonstrates H4R3me2a meeting positive influence cocaine habituation related gene Cdk5 and CaMK II expression,
Illustrate that the compounds of this invention SKLB-639 can control by suppressing cocaine habituation related gene Cdk5 and CaMK II expression
Treat cocaine habituation.
To sum up, the compounds of this invention SKLB-639, it is a kind of PRMT1 inhibitor of high activity, H4R3me2a can be lowered
Expression, reduce gene C dk5 and CaMK II expression, and then suppress cocaine award behavior, treat cocaine habituation, can
To prepare the medicine for turning into treatment cocaine habituation, potential applicability in clinical practice is good.
Claims (9)
- A kind of 1. compound, it is characterised in that：Its structural formula is as follows：。
- 2. purposes of the compound described in claim 1 in PRMT1 inhibitor is prepared.
- 3. compound described in claim 1 is being prepared by reducing H4R3me2a expressions to treat the medicine of cocaine habituation In purposes.
- 4. purposes according to claim 3, it is characterised in that：The medicine is to reduce gene C dk5 and CaMK11 expression water Flat medicine.
- A kind of 5. medicine for treating dependence producing drug habituation, it is characterised in that:It is using compound described in claim 1 as activity into Point, the preparation being prepared plus pharmaceutically acceptable auxiliary material.
- 6. medicine according to claim 5, it is characterised in that:The preparation is ejection preparation.
- 7. purposes of the medicine of claim 5 or 6 in PRMT1 inhibitor is prepared.
- 8. purposes of the medicine of claim 5 or 6 in the medicine for preparing treatment cocaine habituation.
- 9. purposes according to claim 8, it is characterised in that：The medicine is the medicine for reducing H4R3me2a expressions Thing.
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| CN105125571A (en) * | 2015-07-17 | 2015-12-09 | 四川大学 | Application of PRMT1 (protein arginine N-methyltransferase 1) inhibitor to preparation of drug for treating cocaine addiction |
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| CN107375257A (en) * | 2017-08-31 | 2017-11-24 | 同济大学 | Medicinal applications of the inhibitor AMI 1 of arginine methylated transferase in fibrotic disease is suppressed |
| CN108118091B (en) * | 2017-11-22 | 2020-12-15 | 宁波大学 | Kit for detecting methylation degree of PRMT6 gene promoter region related to colorectal cancer and application thereof |
| CN108181461A (en) * | 2017-12-29 | 2018-06-19 | 上海嘉因生物科技有限公司 | Applied to histone modification chromosome co-immunoprecipitation optimization method in tissue samples |
| CN113893346B (en) * | 2020-06-22 | 2022-11-18 | 四川大学华西医院 | Application of GCS inhibitor in preparation of drug for treating cocaine addiction |
| CN113913424B (en) * | 2020-07-09 | 2023-08-08 | 四川大学华西医院 | Adeno-associated virus and application thereof in preparation of medicines for treating cocaine addiction |
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| CN105125571B (en) * | 2015-07-17 | 2018-11-06 | 四川大学 | Purposes of the PRMT1 inhibitor in the drug for preparing treatment cocaine habituation |
Also Published As
| Publication number | Publication date |
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| CN105255887B (en) | 2018-05-18 |
| CN105153042A (en) | 2015-12-16 |
| CN105125571A (en) | 2015-12-09 |
| CN105125571B (en) | 2018-11-06 |
| CN105255887A (en) | 2016-01-20 |
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