CN110372499A

CN110372499A - CARM1 micromolecular inhibitor and its application

Info

Publication number: CN110372499A
Application number: CN201910416650.7A
Authority: CN
Inventors: 赵�权; 鞠君毅
Original assignee: Nanjing Sino Australian Institute Of Translational Medicine Co Ltd
Current assignee: Nanjing Sino Australian Institute Of Translational Medicine Co Ltd
Priority date: 2019-05-20
Filing date: 2019-05-20
Publication date: 2019-10-25
Anticipated expiration: 2039-05-20
Also published as: CN110372499B

Abstract

The invention discloses a kind of CARM1 micromolecular inhibitor and its applications.The micromolecular inhibitor effectively can inhibit prostate gland cancer cell to be proliferated.CARM1 micromolecular inhibitor provided by the invention is low to normal cell toxicity, and there is significant ground inhibiting effect to the CARM1 histone H 3 R17me2a modification mediated and its expression of downstream target gene PSA, therefore tumor cell proliferation can effectively be inhibited, to be expected to be developed into new anti-tumor drug.There is the therapeutic effect of inside and outside to prostate cancer, non-small cell lung cancer, breast cancer, acute and chronic leukemia, liver cancer, gastric cancer, colorectal cancer, oophoroma, cervical carcinoma.

Description

CARM1 micromolecular inhibitor and its application

Technical field

The invention belongs to field of medicinal chemistry, are related to CARM1 inhibitor, and in particular to a kind of CARM1 micromolecular inhibitor And its application.

Technical background

18,000,000 newly-increased cases of cancer are had more than every year according to global cancer statistical result showed in 2018, wherein prostate Cancer disease incidence in all male cancers is number two position, seriously endangers men's health and life.The early treatment of prostate cancer Means include operation excision, radiotherapy and endocrinotherapy etc., but Most patients finally will appear recurrence, develop to go Gesture repellence prostate cancer.Therefore, exploitation low toxicity, efficient and high specific new type antineoplastic medicine become contemporary scientific men Focus on research direction.

1 (coactivator-associated arginine of co-activation factor correlation arginine methyltransferase Methyltransferase 1, CARM1) also it is (the Protein Arginine of arginine methyltransferase 4 Methyltransferase4, PRMT4) be arginine methyltransferase family one of important member.CARM1 nucleus with It is distributed in cytoplasm, the 2nd, 17 and 26 arginine of histone H 3 can be catalyzed, the double methylation modifications of asymmetry occur, In addition there are a variety of nonhistones substrates.CARM1 takes part in a variety of lifes such as transcriptional control, RNA processing, autophagy and Apoptosis Object process.Multiple studies have shown that the imbalance of CARM1 is one of the reason of kinds cancer occurs, height expression and prognosis mala It is related to low survival rate.Therefore, CARM1 has been known as important target for cancer therapy.In the past decade, research reports The micromolecular inhibitor of some CARM1, however lower cell activity and poor specificity kill these small molecules The effect of cancer cell is not satisfactory.Therefore a kind of brand new is researched and developed, small point of natural products with good specificity Sub- compound, by inhibiting the activity of CARM1 to achieve the effect that kill cancer cell, so that developing new anticancer drug has Important realistic meaning and economic value.

Summary of the invention

The purpose of the present invention is to provide the CARM1 micromolecular inhibitor (1a, 1b) of two kinds of enantiomers each other, medium and small point Sub- inhibitor 1b has the function of being able to suppress prostate gland cancer cell proliferation.

Another object of the present invention is to provide above-mentioned CARM1 micromolecular inhibitor answering in the preparation of antitumor drugs With.

Another object of the present invention is to provide a kind of antineoplastic pharmaceutical compositions.

To achieve the above object of the invention, the technical solution adopted by the present invention is that:

Two kinds of CARM1 micromolecular inhibitors (1a, 1b), the inhibitor are the compound with following chemical structures:

Above-mentioned CARM1 micromolecular inhibitor application in preparation of anti-tumor drugs is claimed simultaneously in the present invention.It is excellent Choosing, the tumour is prostate cancer, non-small cell lung cancer, breast cancer, acute and chronic leukemia, liver cancer, gastric cancer, Colon and rectum Cancer, oophoroma, any one in cervical carcinoma.

The CARM1 micromolecular inhibitor for the brand new that the present invention develops, which mainly passes through, inhibits CARM1 transmethylase living Property, entirety histone H 3 R17 methylation modification level is reduced, prostate-specific antigen (PSA) gene is inhibited Expression, so as to effectively inhibit the effect of tumor cell proliferation.Therefore, the present invention can answer in preparing anti-tumor drug With.

A kind of antineoplastic pharmaceutical compositions, the active constituent packet of the antineoplastic pharmaceutical compositions is claimed in the present invention simultaneously Containing above-mentioned CARM1 micromolecular inhibitor.Preferably, the antineoplastic pharmaceutical compositions further include pharmaceutically receptible carrier or Auxiliary material.

CARM1 micromolecular inhibitor of the present invention can be made individually or with more than one acceptable carrier combination agents Preparation administration.For example, solvent, diluent etc..It can be with oral dosage form, such as tablet, capsule, dispersible powder, granule； It can also be administered with injection-type, such as freeze drying powder injection.The various dosage forms of pharmaceutical composition of the invention can be according in pharmaceutical field Well known method preparation.

Due to the application of the above technical scheme, the present invention has compared with prior art with following advantages:

1, CARM1 micromolecular inhibitor prepared by the present invention, obtains from the Separation of Natural Products of plant, can use biology The preparation of the method for synthesis or microbial fermentation reacts simple to operation, and product is easily purified, yield is high, easy to maintain.

2, the CARM1 micromolecular inhibitor that the present invention is identified is low to normal cell toxicity, and to CARM1 substrate histone The expression for the downstream gene PSA that H3R17 methylationization mediates has significant inhibiting effect, therefore can effectively inhibit forefront The proliferation of gland cancer, to be expected to be developed into new anti-tumor drug.

Detailed description of the invention

Fig. 1 1a's and 1b¹H nuclear magnetic resonance spectroscopy (500MHz, DMSO-d6) result

Fig. 2 1a's and 1b¹³C nuclear magnetic resonance spectroscopy (125MHz, DMSO-d6) result

Fig. 3 1a's and 1b¹H-¹H relevance magnetic vibration spectrum analysis (DMSO-d6) result

Fig. 4 chiral high performance liquid chromatography separates 1a and 1b

Fig. 5 methylating in vitro experimental analysis micromolecular inhibitor 1a and 1b inhibit the effect of CARM1 enzyme activity in vitro

The dissociation constant of Fig. 6 MST technology detection 1b small molecule and CARM1 albumen

Fig. 7 protein immunoblot studies influence of the 1a and 1b to CARM1 substrate H3R17me2a level

Fig. 8 protein immunoblot studies influence of the 1a and 1b to CARM1 expression

The effect of CARM1 interference expression in Fig. 9 real-time quantitative PCR and protein immunoblot verifying LNCap cell

The expression of PSA after CARM1 interference is expressed in Figure 10 real-time quantitative PCR and protein immunoblot verifying LNCap cell Variation

Figure 11 real-time quantitative PCR and protein immunoblot verifying 1a and 1b handle the expression change of PSA after LNCap cell respectively Change

Influence of Figure 12 1a and 1b to the LNCap cell cycle

Influence of Figure 13 1a and 1b to LNCap total protein of cell

Figure 14 protein immunoblot is verified wild type CARM1 and Inactivating mutations CARM1 and is overexpressed in LNCap stable cell line The expression of CARM1

Figure 15 CCK-8 experiment detection 1a and 1b is overexpressed LNCap to wild type CARM1 and Inactivating mutations CARM1 and stablizes carefully The influence of born of the same parents' strain proliferation

Specific embodiment

The invention will be further described for Binding experiment:

1 1a of embodiment, 1b preparation method:

Natural products 1a, 1b derive from the enterobacteriaceae IFB-TL01 of mantis (Tenodera aridifolia) (in being preserved in State's Type Tissue Collection, deposit number CCTCCM207198), by IFB-TL01 after being separated in mantis enteron aisle, in vitro Mass propgation will pass through fractional method (the Kruzselyi D et al.Journal based on bioactivity after bacterium ultrasonication Of Chromatographic Science, 2016,54 (7): 1084-1089.) a kind of isolated life with completely new skeleton Object activity polyketide, high-resolution electrospray ionization mass spectrometry is analysis shows that compound [the M+Na]+charge-mass ratio is 541.0897.We further pass through¹H and¹³C NMR,¹H-¹H COSY analyzes to determine its structure (Fig. 1-3), Single Crystal X-ray Crystallographic analysis shows that the compound is the racemic mixture formed by a pair of of enantiomer.Further we pass through efficient liquid Phase chromatography is separated (Fig. 4) to two kinds of chiral enantiomers, they are named as 1a and 1b by us.

Extracellularly influence of the experiment detection 1a and 1b to CARM1 catalytic activity of embodiment 2

In order to verify 1a and 1b it can inhibit the methyl transferase activity of CARM1, prokaryotic expression has GST first for we The CARM1 albumen of label, has carried out methylating in vitro experiment, and shown in Fig. 5, GST-CARM1 albumen has methyl transferase activity, Can methylate histone H 3.In addition, be compared with a control, 1a and 1b can be significant inhibit CARM1 methyl transferase activity, And 1b effect is more preferable.

Then we move the combination situation of instrument (MST) detection 1b and CARM1 albumen using micro thermophoresis.MST is to utilize grain Directed movement of the son in microcosmic temperature gradient, by measurement hydrated sheath (usually by the variation of biomolecular structure/conformation It is caused) caused by micro thermophoresis dynamic variation determine affinity.Shown in Fig. 6, vitro binding assay shows 1b and CARM1 Albumen is bound directly, and binding constant is 40.75 μM ± 3.05 μM.

Specific step is as follows:

A.GST-CARM1 prokaryotic protein expression

1) construction of recombinant plasmid: we select pGEX-6p-1 vector construction pGEX-6p-1-CARM1 prokaryotic expression carrier. Using EcoR I and Sal I restriction enzyme site, PCR primer is as follows:

Using people 293T cell cDNA as template, LATaq enzymatic amplification CARM1 CDS sequence, double digestion amplified fragments, pGEX- 6p-1 carrier carries out recombinant plasmid connection, and connection product converts DH5 α competence bacteria, smears the LB plate of the resistance of benzyl containing ammonia, chooses Positive colony is taken to carry out sequencing identification, it is pGEX-6p-1-CARM1 group plasmid that correct plasmid, which is sequenced,.

2) prokaryotic protein expression: positive recombinant plasmid pGEX-6p-1-CARM1 conversion BL21 competence is expressed into strain, is connect 100 μ l prokaryotic expression bacterium are taken to cultivate into 3ml LB culture medium.Expand culture by 1:100 volume ratio within second day, is taken after 3 hours 1ml bacterium solution surveys OD600 light absorption value, and after OD600 value reaches 0.4-0.6, IPTG (1M) induction is added according to 1:10000 ratio CARM1 prokaryotic protein expression, after continuing culture 4 hours, thalline were collected by centrifugation by 10000rpm, -80 DEG C of preservations.

3) GST-CARM1 protein purification: thallus is resuspended with 1L bacterium/50ml PBS ratio, is placed in cooled on ice, places ultrasound In broken instrument, thallus is crushed with following procedure: power 600w, ultrasonic 2s, interval 2s, total 30min.After ultrasound 12000rpm, 4 DEG C of centrifugation 15min removal precipitatings, collects supernatant into new 50ml pipe, and 300ml GST beads, 4 DEG C of rotations are added Turn to combine 1 hour, 3000rpm, 4 DEG C centrifugation 3min, remove supernatant, washed 3 times using 2ml NETN500 solution, is taken appropriate GST beads carries out SDS-PAGE electroresis appraisal purification result.

4) GST-CARM1 prokaryotic recombinant protein elutes: reduced glutathione is prepared according to the following formulation elutes solution:

500 μ l of 1M Tris-Cl (pH=8.0)

DdH2O to 10ml

L-glutathione 0.03g

1ml eluent is added into the EP pipe of the beads containing GST, 4 DEG C of rotations 30min, 3000rpm are centrifuged 3min, draw Supernatant retains, and elutes 2 times repeatedly, and the supernatant of reservation is GST-CARM1 solution, -80 DEG C of preservations.Take appropriate supernatant Liquid carries out SDS-PAGE electroresis appraisal elution effect, purity of protein and protein quantification.

B. methylating in vitro reacts

Methylating in vitro reaction is carried out by following system:

After mixing each ingredient by above-mentioned system, 30 DEG C of reaction 2h are placed in, carry out autoradiograph experiment after reaction.

C. minute yardstick thermophoresis experiment (MST experiment)

We use the instrument of MONOTEMPER company by MST experimental verification 1b and CARM1 albumen direct interaction With kit (article No.: MO-C030), steps are as follows for specific experiment:

A) buffer-exchanged

Requirement according to MST instrument to test sample, marking operation need protein dissolution in the label of neutral pH (pH6-8) In buffer, primary amine compound (such as ammonium ion, Tris, glycine, ethyl alcohol cannot be contained in proteolytic buffer Amine, triethylamine, glutathione) or imidazoles, this kind of compound can be substantially reduced protein labeling efficiency.Purity of protein it is lower or The carrier protein contained in protein sample such as BSA can influence the label of albumen, therefore we are first, in accordance with the step in kit Suddenly buffer-exchanged is carried out to CARM1 albumen, concrete operations are as follows:

1) add the buffer salt in 3ml distilled water solubilising reagent box.

2) it is inverted and mixes column A, twist off the small lid of the bottom column A, and unscrew column lid.

3) pillar is placed in the EP pipe of 1.5-2ml, 3000rpm is centrifuged 1min and removes excessive liquid in column A, is added The 1 of 300 μ l) in the buffer that dissolve, 3000rpm centrifugation 1min, washing 3 times.

4) column A is added in the protein solution of 40-100 μ l, column A is put into new EP pipe, 3000rpm, 4 DEG C of centrifugations 2 Min to get exchange buffering liquid albumen.

B) label of albumen

1) concentration of albumen is adjusted to 2-20 μM with label buffer.

2) the DMSO dissolved solid dyestuff of 50 μ l is added (concentration of dyestuff is about 650 μM at this time).

3) it mixes, so that dyestuff sufficiently dissolves, the concentration with label buffer dilution dyestuff is 2-3 times of protein concentration.

4) 1:1 volume mixture dyestuff and albumen, room temperature, which is protected from light, is incubated for 30min.

C) purifying of albumen

In order to optimize the experimental result of MST, unreacted excess free dye needed column to remove, the purity of labelled protein Can by measurement albumen and dyestuff ratio obtain (such as can by measurement albumen in 280nm and dyestuff at 650nm Absorbance value come the ratio that both measures, molar absorptivity 250M-1cm-1).

1) storing liquid in column B is outwelled, (8ml is needed in total, by buffer under gravity from column with MST test fluid balance columns B Balance pillar is flowed out in B).

2) the label reaction solution of 200 μ l volumes is added to the middle position column B, allows reaction solution to be completely immersed in column B, discards stream Liquid out.

3) flushing liquors (kit offer) of 300 μ l volumes is added to column B, repetitive operation 2.

4) flushing liquor of 600 μ l volumes is added, collects the liquid eluted (liquid that preceding two drip goes out can discard).

5) ratio of spectroscopy measurements albumen and dyestuff is used, and dispenses albumen.

D) MST reacts

1b small molecule mother liquor is taken, is successively diluted with 2 times, dilutes 16 PCR pipes altogether, every pipe is added in c with 1:1 volume and is marked Albumen, mix, drawn using capillary at the top of capillary, avoiding bubbles inside capillary.Capillary is put into Nano Temper MST instrument reads fluorescent value, calculates dissociation constant KD value using instrument software program.

Influence of the detection 1a and 1b to CARM1 catalytic activity into the cell of embodiment 3

In order to verify the influence of 1a and 1b to CARM1 catalytic activity in the cell, we handle LNCap using 1a and 1b Cell collects cell after 48h, extracts albumen and carries out protein immunization imprinting experiment, uses different histidine tag antibody tests The variation of associated histones modification, 1a and 1b processing LNCap cell reduces H3R17me2a modification water to Fig. 7 significantly as the result is shown It is flat, but it is horizontal not influence H4R3me2a and H4R3me2s modification.Fig. 8 is not the result shows that 1a and 1b influence the albumen water of CARM1 It is flat.Therefore, above-mentioned experimental result confirms that 1a and 1b can inhibit the intracellular H3R17me2a modification of LNCap horizontal, and 1b table Reveal stronger inhibitory effect.

Embodiment 4 constructs the LNCap stable cell line of CARM1 interference expression, detection GAP-associated protein GAP variation

In order to verify function of the CARM1 in prostate cancer, we construct CARM1 using shRNA slow virus system and do Disturb the LNCap stable cell line of expression.Real-time quantitative PCR and protein blot have detected CARM1 expression (Fig. 9), as a result table Bright CARM1mRNA and protein expression level all decline, while the experiment of real-time quantitative PCR protein blot also confirms that CARM1 interference table After reaching, the expression of prostate cancer specific antigen PSA significantly reduces (Figure 10).The above results show that CARM1 can regulate and control The expression of PSA in prostate cancer.

Specifically embodiment and steps are as follows:

1) building and detection of the prostate cancer LNCap stable cell line of CARM1 interference expression

The LNCap stable cell line that we are expressed using the building CARM1 interference of pLL3.7shRNA slow virus system. LNCap cell is bought from Shanghai cell biological, the training in the DMEM culture medium (V/V, Thermofisher) containing 10%FCS It supports.The shRNA target sequence of CARM1 is inserted into the position XhoI/HpaI of pLL3.7 slow virus plasmid in accordance with the specification of manufacturer Point.ShRNA target spot:

2) using Real-time quantitative PCR detection CARM1 gene in control scr cell and CARM1 interference expression cell strain (CARM1-kd) the mRNA expression in, specific implementation step are as follows:

A. cell total rna extracts

1) tally method checks that cell density (takes surrounding and intermediate grid to count, calculates average value, it is desirable that cell density 5 × 106/ml).

2) in Biohazard Safety Equipment, 5ml cell solution is taken to be added in sterile 15ml centrifuge tube, 1000rpm centrifugation 10min。

3) supernatant is removed, 1ml PBS is added, blows even cell, these cell solutions are fully transferred in an EP pipe (following institute EP pipe, pipette tips must pass through 0.1%DEPC water process, use after moist heat sterilization).

4) supernatant is removed, 1ml PBS is added, blows even cell, 3000rpm 5min.

5) exhaust supernatant, discard, respectively plus 1ml Trizol, often plus a pipe, must piping and druming uniformly, be subject to and lose filiform (i.e. transparent), is stored at room temperature 5min.

6) 200 μ l chloroforms are added, 15s is aggressively shaken, is stored at room temperature 2-3min.

7) 12000g, is centrifuged 15min by 4 DEG C.

8) upper strata aqueous phase (about 600 μ l) is drawn, is transferred in a new EP pipe, adds 500 μ l isopropanols, be stored at room temperature 10min。

9) 12000g, is centrifuged 10min by 4 DEG C.

10) supernatant is removed, adds the pre-cooled ethanol (0.1% DEPC water is prepared) of 1ml 75%, is vortexed a moment.

11) 7500g, is centrifuged 5min by 4 DEG C.

12) supernatant is removed, it is dry, it is dissolved in the 0.1%DEPC water of 20 μ l, 55 DEG C of placement 10min dissolutions utilize spectrophotometric Meter measurement concentration and purifying, RNA can be immediately available for reverse transcription cDNA or -80 DEG C save backup.

B.cDNA synthesis

1) HiScriptRverse Transcriptase Transcription System

Mixed liquor is formulated as follows in the EP pipe of no RNase:

Mixing, 42 DEG C of 2min are gently blown and beaten with pipettor.5 × qRTSuperMix II is added later, gently with pipettor Piping and druming mixes, and synthesizes cDNA by following procedure:

25℃ 10min

50℃ 30min

85℃ 5min

Product can be immediately available for PCR reaction, or save backup at -20 DEG C.

Q-RT-PCR uses 6000 quantitative PCR apparatus of Rotor-gene of Gene company Corbett Research, examination Agent is Roche FastStart Universal SYBR Green Master Mix, and reaction system is as follows:

Q-RT-PCR reaction condition is as follows:

2) CARM1, PSA list of primers are as follows:

Embodiment 5 1a and 1b is to PSA expression, the influence of cell cycle and total protein in LNCap cell

We handle LNCap cell using 1a and 1b respectively, extract cell total rna and total protein of cell, detect PSA base The expression of cause.Real-time quantitative PCR is the results show that relative to control cell, and PSA gene expression dose is significantly reduced, and albumen is exempted from Epidemic disease Blot results also confirm that PSA protein level reduces (Figure 11), this result is identical as CARM1 interference expression effect.

In order to further analyze the influence of 1a and 1b to LNCap cell, we used cell cycle detection kits.Stream Formula Cytometric Analysis the result shows that, 1b handle LNCap cell after, cell-cycle arrest is at G1 phase (Figure 12).By to cell Total protein carries out the analysis of SDS-PAGE electrophoresis dying, the results showed that 1a and 1b processing LNCap cell by cell total protein levels do not have Have a significant impact (Figure 13).

Embodiment 6 1a and 1b inhibit the detection of tumor cell proliferation ability

By cancer cell LNCap, A549, MCF7 and normal cell 293T are passaged in 96 orifice plates, extremely to cell adherent growth After logarithmic growth phase, the 1a of various concentration and (0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM) of 1b are added Enter into the cell in 96 orifice plates, 37 DEG C of culture 48h, analyze cell growth status with CCK-8 method, calculates 1a and 1b to difference The half-inhibitory concentration IC of cell₅₀.Table 1 shows that 1b has good inhibiting effect to prostate cancer LNCap cell Proliferation.

Table 1 1a and 1b handles the half-inhibitory concentration IC of different cells₅₀

Cell lines	LnCap	A549	293T	MCF7
					1a	>14μM	>14μM	>14μM	>14μM
1b	9μM	>14μM	12.81μM	12.74μM

The building and identification of embodiment 7 wild type CARM1 and Inactivating mutations CARM1 overexpression LNCap stable cell line

It is to inhibit LNCap cell Proliferation by inhibiting CARM1 catalytic activity to verify 1a and 1b in cellular level.I Construct MSCV-CARM1 over-express vector (the concrete operation step bibliography Nie M et with HA label first Al.Journal of Biological Chemistry, 2018,293 (45): 17454-17463.), then pass through point mutation Kit (SBS Genetech, article No. SDM-15) is mutated on the basis of MSCV-CARM1 over-express vector and has obtained MSCV-CARM1 S229E Inactivating mutations over-express vector.Transfected jointly with packaging plasmid 293T cell packaging be overexpressed MSCV-CARM1 and The retrovirus of MSCV-CARM1S229E infects LNCap cell, constructs the LNCap that CARM1 and CARM1S229E is overexpressed Stable cell line (concrete operation step bibliography Nie M et al.Journal of Biological Chemistry, 2018,293(45):17454-17463.).Carrying out protein immunoblot experiment using HA antibody proves that cell strain constructs successfully (Figure 14).Then we handle what wild type (WT) and Inactivating mutations (S229E) CARM1 were overexpressed using 1a and 1b respectively LNCap cell, by CCK-8 test detection ability of cell proliferation variation, Figure 15 the result shows that 1a and 1b for wild type The inhibitory effect for the LNCap ability of cell proliferation that CARM1 is overexpressed is significant, and to the LNCap that Inactivating mutations CARM1 is overexpressed The inhibitory effect of ability of cell proliferation is poor.This is the result shows that 1a and 1b are played by inhibiting the catalytic activity of CARM1 Effect.

Claims

1.CARM1 micromolecular inhibitor, which is characterized in that the inhibitor is the chemical combination with the chemical structure as shown in 1a or 1b Object:

2. CARM1 micromolecular inhibitor application in preparation of anti-tumor drugs described in claim 1.

3. application according to claim 2, which is characterized in that the tumour is prostate cancer, non-small cell lung cancer, cream Gland cancer, acute and chronic leukemia, liver cancer, gastric cancer, colorectal cancer, oophoroma, any one in cervical carcinoma.

4. a kind of antineoplastic pharmaceutical compositions, which is characterized in that the active constituent of the antineoplastic pharmaceutical compositions is wanted comprising right CARM1 micromolecular inhibitor described in asking 1.

5. antineoplastic pharmaceutical compositions according to claim 4, which is characterized in that the antineoplastic pharmaceutical compositions further include Pharmaceutically receptible carrier or auxiliary material.