CN102697762A - Application of parietic acid or parietic acid compound in preparing drug for treating disease taking FTO (Fat mass and obesity-associated) as target point - Google Patents

Abstract

The invention relates to an application of a parietic acid or parietic acid compound expressed by a formula I in preparing a drug for treating a disease taking FTO (Fat mass and obesity-associated) as a target point. An experimental result shows that the compound expressed by the formula I can specifically combine with FTO; the restraining activity is expressed by competing for a substrate binding site; and an intracellular test also shows that the level of N-6 methylation adenine (6meA) in messenger RNA (Ribonucleic Acid) in vivo is obviously increased by the compound expressed by the formula I.

Description

Chrysophanic acid or rhein compound are the purposes in the medicine of disease of target spot with FTO in preparation treatment

Technical field

The present invention relates to field of pharmacology, relating in particular to chrysophanic acid or rhein compound is the purposes in the medicine of disease of target spot with FTO in preparation treatment.

Background technology

Fat relevant FTO (Fat mass and obesity-associated) gene mapping is in human No. 16 chromosomes, its protein product wide expression in human body.FTO genovariation and obesity show bigger relatedness, so this gene is called " ob gene " (Frayling TM, et al., Science, 2007316 (5826): 889-94 again by people; Gerken T, et al., Science, 2007,318, (5855): 469-72).The nucleic acid oxidation demethylase AlkB family protein that FTO and Alpha-ketoglutaric acid relies on has higher sequence homology.FTO can the catalysis single stranded deoxyribonucleic acid N-3 position thymus pyrimidine (3meT) and the middle N-3 position of ribonucleic acid (RNA) uracil (3meU) the oxidation demethylation that methylates that methylates in (DNA).Research recently shows, in experiment in vitro, the N-6 position adenine (6meA) that methylates is that of FTO mainly acts on substrate (Jia G among single stranded DNA and the RNA; Et al., Nat.Chem.Biol., 20117 (12): 885-7); In vivo, the messenger RNA that contains (6meA) is the effect substrate of FTO.

So far people are not clear to the relation of the function of FTO and disease.Existing research shows that FTO can ingest to the mankind and have a direct impact, also maybe be relevant with Alzheimer's disease, metabolism syndrome etc. (Obesity 2,008 16 (8): 1961-5 for Speakman JR, et al.; Keller L, et al., J Alzheimers Dis.2011 23 (3): 461-9; Freathy RM, et al., Diabetes 200857 (5): 1419).FTO and human health are in close relations, thereby the function of FTO also is the focus of studying both at home and abroad with regulation and control always.Yet, do not see report at present relevant for the FTO inhibitor.The discovery of FTO inhibitor will be to the research of FTO function and the new drug development generation significant impact that is directed against the FTO target spot.

Summary of the invention

Technical problem

Also checking is to the micromolecular compound inhibitor of FTO in design, and it has and suppresses FTO and remove the N-6 position methylated biological activity in the adenine (6meA) that methylates.

Technical scheme

In order to quicken designing and screening FTO inhibitor; The inventor at first uses high flux virtual screening (In silico HTS) technology; Promptly through the analysing protein three dimensional structure; By computer program and analysis software, filter out the chemical compound that major part does not meet the demands in the compound library, screening has obtained 141 candidate compounds from 280,000 chemical compounds.

Obtain having the active micromolecular compound of inhibition through external FTO biological activity confirmatory experiment.Show the inhibition activity in order to get rid of chemical compound owing to destroying secondary protein structure, the inventor has used circular dichroism spectra (CD) technology.Far ultraviolet CD is one of main means of research protein secondary structure in solution.Alpha-helical structure shows two minus characteristic acromion bands of a spectrum in 222 nanometers and 208 nanometers; Can utilize the molar ellipticity [θ] 208 at this two place or the content that [θ] 222 estimates Alpha-spiral in the protein, judge whether secondary structure acute variation has taken place.

For study reactive compound whether can with the FTO protein bound, the inventor verifies through differential scan fluorescence (DSF).Gibbs free energy changeization in proteinic stability and the unfolding process (△ Gu) has certain dependency, and is that temperature relies on.Most of proteic stability changes with temperature, and when temperature raise, △ Gu reduced, when folding albumen with unfolding reaches balance, and △ Gu=0, the temperature of this moment is defined as proteinic thaw temperature (Tm).If proteinic unfolding carries out with reversible bifurcation mode, the equilibrium state thermodynamic model will be applicable to this process so.If chemical compound can with protein bound, the bonded Gibbs free of chemical compound in most cases can cause △ Gu to increase, corresponding Tm value rising.The fluorescence signal of fluorescent dye Sypro Orange is gone out by collection in water environment, and along with the rising of temperature, the protein unfolding exposes hydrophobic region, and Sypro Orange is bonded to protein hydrophobic region, emitting fluorescence.Fluorescent value is done curve to temperature, and the temperature of the transition state position of protein unfolding is defined as Tm.Research shows that the protein stability raising that chemical compound combines to cause is directly proportional with compound concentrations and affinity.

Then, the inventor through biomolecular interaction analysis (BIA) technology assessment chemical compound and bonded specificity of FTO and intensity.BIA is based on the biosensory analysis technology of this physical optics phenomenon of surface plasma resonance (SPR)., incident illumination will produce total reflection when inciding the interface of two kinds of different transparent mediums with critical angle; And intensity of reflected light all should be identical on all angles; But if after dielectric surface plates the layer of metal thin film; Because incident illumination can cause the resonance of free electron in the metal, thereby causes reflected light in certain angle, to weaken greatly, wherein makes the angle of reflected light complete obiteration be called resonance angle.The change of the refractive index of the liquid phase that resonance angle can pass through with the metallic film surface changes, and change of refractive is directly proportional with the macromole quality that is combined in the metal surface again.BIA is exactly the change that utilizes the refractive index on metallic film surface, causes the variation of resonance angle, infers the variation on metallic film surface.Earlier a kind of biomolecule is fixed on sensor chip surface during experiment, interactional molecule is dissolved in flow of solution and crosses chip surface with it.Owing to needn't use fluorescent labeling and isotopic labeling, thereby keep the natural activity of biomolecule.Detector can be followed the tracks of the variation of the molecule (like chemical compound) that detects in the solution and the whole process that combines, dissociates of chip surface molecule (like protein).Can observe two kinds of bonded specificitys of molecule through it, can know that how strong being combined with of two kinds of molecules be.

In order further to study the model of action of chemical compound and FTO, the inventor has used isothermal titration calorimetric (ITC) technology.The ITC technology is a kind of postgraduate's thermodynamic and the dynamic (dynamical) important method that development in recent years is got up; It monitors and writes down the calorimetric curve of a change procedure continuously, exactly through the micro-calorimeter of high sensitivity, high automation, original position, online and thermodynamics and kinetics information is provided with no damage simultaneously.The complete information of a whole set of relevant interaction of molecules can be provided in single experiment.ITC is one of classical way of characterising biological interaction of molecules, is widely used in studying the interaction of micromolecular compound and biomacromolecule (protein, nucleic acid etc.).

Suppress active mode in order to illustrate the chemical compound performance, the inventor has used fluorescence polarization (FP) technology.The ultimate principle of FP be fluorescent material behind the polarizing light irradiation of single plane, absorb luminous energy and leap to excited state, return back to ground state subsequently, and send the polarized fluorescence of single plane.The strong and weak degree of polarized fluorescence and the size of fluorescence molecule are proportionate, and the speed of rotating when being stimulated with it is inverse correlation.With fluorescent labeling FTO substrate DNA, after DNA and protein binding, effectively fluorescence molecule becomes big, and polarized fluorescence strengthens.If chemical compound can influence the interaction of DNA and FTO, fluorescence intensity can corresponding changing.

The inventor studies based on virtual screening and model of action that the FTO crystal structure is carried out micromolecular inhibitor, has found that first chrysophanic acid or rhein compound can suppress the FTO activity, and the structure of this compounds is shown in following general formula I:

Wherein, R ₂~ R ₃, R ₆~ R ₇In have at least one for-COOH ,-COONa ,-COOK ,-SO ₃H ,-SO ₃Na ,-SO ₃K; R ₁～R ₈All the other positions be following substituent group :-H ,-OH ,-NH ₂, halogen.

Chrysophanic acid shown in the above-mentioned general formula I or rhein compound also comprise the salt that itself and alkali metal form, for example sodium salt, potassium salt etc.

Because chrysophanic acid or rhein compound shown in the general formula I can suppress the FTO activity, it can be used to prepare treatment is the medicine of the disease of target spot with FTO.

Chrysophanic acid of the present invention or rhein compound can be according to the method preparations of routine.For example, chrysophanic acid and sodium salt thereof and potassium salt can prepare through following method in the general formula I:

Get chrysophanol 6g, add the mixed liquor of 150ml acetic anhydride and pyridine (1:1 volume ratio), ambient temperature overnight, reactant is poured crystallization in the cold water into, filters oven dry; Be added in the mixed liquor of 300ml acetic anhydride and glacial acetic acid (1:1 volume ratio), 45 ℃ drip chromic acid solution, and 65 ℃ were stirred 8 hours, and reactant is poured in the water, crystallization; Filter, add 1 liter of 25% sodium carbonate liquor, chloroform extraction 3 times boils sodium carbonate liquor, cooling; Add hcl acidifying, treat that gas arrange after, heated and boiled 1 hour is cooled off crystallization; Sucking filtration, washing, the glacial acetic acid recrystallization gets the 2g chrysophanic acid, and purity is more than 98%.Chrysophanic acid sodium salt, potassium salt are transferred pH to 8.0 preparation with NaOH and KOH respectively.

Chrysophanic acid of the present invention or rhein compound can also extract preparation according to the method for routine.For example, chrysophanic acid and sodium salt thereof and potassium salt can obtain through following method in the general formula I:

Get Radix Et Rhizoma Rhei powder 500g, add 5 times of amount 60% alcohol heating reflux 2 times, each 1 hour, merge extractive liquid, was concentrated into about 1000ml; Add the 100ml concentrated hydrochloric acid, 50 ℃ of hydrolysis 1 hour, cooling is filtered, and in deposition, adds 1000ml 5%NaHCO3 solution; Heating for dissolving is filtered, and adds ethanol to 50～90% in the filtrating, filters, and filtrating is transferred below the pH to 3 with concentrated hydrochloric acid; Cross and filter yellow mercury oxide, washing is to neutral, and the glacial acetic acid recrystallization gets chrysophanic acid, and content is more than 98%.Chrysophanic acid sodium salt, potassium salt are transferred pH to 8.0 preparation with NaOH and KOH respectively.

Beneficial effect

The inventor uses the research means of biochemistry and biophysics, has disclosed the model of action of compound of Formula I and FTO, and experimental result shows that the chemical compound shown in the general formula I can combine with FTO specifically, suppresses active through the performance of competition substrate binding site.Test also shows compound of Formula I the methylate level of adenine (6meA) of N-6 position in the messenger RNA that can obviously raise in the body in the cell.

Description of drawings

Fig. 1 is the chemical compound high flux virtual screening figure based on structure;

Fig. 2 does not cause secondary protein structure significant change figure for CD technology proof chemical compound;

Fig. 3 is that DSF technology proof chemical compound can combine figure with FTO;

Fig. 4 is BIA technical Analysis chemical compound and bonded specificity of FTO and intensity map;

Fig. 5 is the bonded thermodynamics and kinetics figure of ITC characterized by techniques chemical compound and FTO;

Fig. 6 can compete binding site figure with nucleic acid primer for FP technology proof chemical compound;

Fig. 7 can make 6meA concentration rising figure in BE (2)-C cell mRNA for chemical compound.

The specific embodiment

Below with reference to accompanying drawing exemplary embodiments of the present invention is described, to help to understand the present invention.

Embodiment

EXPERIMENTAL EXAMPLE 1: based on the chemical compound high flux virtual screening of structure.

The structure that virtual screening adopted is the people source FTO (PDB number: 3LFM) that has combined substrate 3meT; Structure is downloaded from Protein Data Bank (http://www.rcsb.org/pdb), obtains removing intramolecular hydrone and buffer and other ions behind the crystal structure.The inventor has screened 280,000 chemical compounds, has bought 114 candidate compounds, and concrete screening process is as shown in Figure 1.

EXPERIMENTAL EXAMPLE 2: the inhibition of biochemical test detection compound is active.

The pET-28a-FTO prokaryotic expression plasmid is converted into BL21 (DE3)-RIL expression strain; 0.5mM IPTG abduction delivering is 17 hours under 16 ℃ of conditions; Collect the thalline ultrasonication; Behind centrifugal 20 minutes of the 15000rpm, get supernatant and behind Ni-NTA, MonoQ and Superdex200 purification, obtain the proteic purity of FTO greater than 95%.In the sieve medicine system of 100 μ l, adding substrate 6meA-RNA concentration is 10 μ M, and the FTO protein concentration is 1 μ M, adds the testing compound of variable concentrations, hatches 1 hour 100 ℃ of 10 minutes deactivation albumen for 25 ℃.Use the RNA behind P1 enzyme and the alkali phosphatase endonuclease reaction then, HPLC analyzes the methylate ratio of adenine (6meA) of adenine (A) and N-6 position.The concentration that enzymatic activity suppression ratio (%) is reached 50% o'clock inhibitor is decided to be IC ₅₀Value.Suppression ratio (%) can be according to computes: suppression ratio (%)={ [experimental group 6meA/ experimental group (A+6meA)]/[matched group 6meA/ matched group (A+6meA)] } * 100%

In 114 candidate compounds, detect demonstration through BA, chrysophanic acid or rhein compound have active to the inhibition of FTO preferably.For further test chrysophanic acid or rhein compound are active to the inhibition of FTO, the application has tested the chemical compound shown in a series of following general formula Is:

Wherein, R ₂~ R ₃, R ₆~ R ₇In have at least one for-COOH ,-COONa ,-COOK ,-SO ₃H ,-SO ₃Na ,-SO ₃K; R ₁~ R ₈All the other positions be following substituent group :-H ,-OH ,-NH ₂, halogen.

The particular compound of structure shown in the general formula I is as shown in table 1.This compounds is active to the inhibition that FTO shows in various degree.Table 2 has provided that part of compounds is to the inhibition active testing result of FTO in the table 1, and wherein, the inhibition activity of 1 couple of FTO of chemical compound is best, IC ₅₀Value is 21 μ M.Compound used therefor is sodium-salt form in the experiment.

Table 1: the particular compound of general formula I structure

Table 2: chemical compound suppresses active to FTO

A is three groups of panel datas, its SD < 10%

EXPERIMENTAL EXAMPLE 3:FTO is the oxidase that 2OG/Fe (II) relies on, and the oxidase that relies on to 2OG/Fe (II) at present is mainly the analog of 2OG or the chelating agen of Fe (II).Micromolecular inhibitor to FTO does not appear in the newspapers as yet.For get rid of chemical compound through complexation Fe (II) just show the inhibition of FTO actively, this paper has at first designed following experiment.Keeping in matched group, adding the Fe (II) excessive under the active impregnable condition of FTO, correspondingly, in the parallel laboratory test group, add 100 μ M chemical compounds 1 than chemical compound.The result shows that the inhibition activity of chemical compound 1 does not rely on the variation of Fe (II) concentration and changes, and the result is shown in table 3.

Table 3: the inhibition of 1 couple of FTO of chemical compound is active under different Fe (II) concentration

A is three groups of panel datas, its SD < 10%

EXPERIMENTAL EXAMPLE 4:CD technology proof chemical compound does not cause the secondary protein structure significant change.

With chemical compound and protein according to mol ratio 50:1 and 100:1 mixing, hatch 20 minutes after, carry out the far-ultraviolet spectrum data collection at 200-250nm, three groups of repetitions are established in experiment.The spectral data analysis shows that chemical compound does not cause the significant change of secondary protein structure, and is as shown in Figure 2.

EXPERIMENTAL EXAMPLE 5:DSF technology proof chemical compound can combine with FTO.

With chemical compound 1 and FTO according to mol ratio 10:1,20:1,40:1 mixing after, add fluorometric reagent SYPRO orange, at AB 7500Fast RT-PCR appearance working procedure: 25 ℃～95 ℃, heating rate is 1%, every first order fluorescence of reading at a distance from 20 seconds.Fluorescent value is done curve to temperature, and through the Boltzmann match, the temperature of the transition state position of protein unfolding is defined as T _mValue, three groups of repetitions are established in experiment.The result shows that along with chemical compound 1 concentration raises, △ Tm value increases.As shown in Figure 3.

Bonded specificity of EXPERIMENTAL EXAMPLE 6:BIA technical Analysis chemical compound and FTO and intensity.

The purity that purification obtains is dialysed to HBS-EP buffer (0.01MHEPES pH 7.4,150mM NaCl, 3mM EDTA, 0.005%v/v Surfactant P20) greater than 95% FTO albumen, and protein concentration is 5mg/ml.Chemical compound 1 and 5 is dissolved as a series of Concentraton gradient with the buffer behind the dialysis albumen respectively.Albumen is fixed in the CM5 chip after being diluted to 0.1mg/ml with sodium acetate buffer (pH 4.2).Ready sample is put into BIAcore 3000 collect data, the result shows, the K of chemical compound 1,4,5 _dBe respectively 49.4 μ M, 11.5 μ M and 8.3 μ M, as shown in Figure 4.

The bonded thermodynamics and kinetics of EXPERIMENTAL EXAMPLE 7:ITC characterized by techniques chemical compound and FTO.

The purity that purification is obtained greater than 95% FTO albumen dialysis to experiment with buffer (50mMTris-HCl, pH 8.0,500mM NaCl, 10mM β-Me), concentration is 65 μ M, volume is 2mL.Chemical compound 1 is dissolved in the buffer behind the dialysis albumen, and concentration is 650 μ M, and volume is 500 μ L.Chemical compound is splashed into protein sample, and every separated 3min drips once totally 25 times.Contrast is adopted and is used the same method, and chemical compound is splashed into buffer.The result shows, chemical compound and FTO are combined into the endothermic reaction, K _dBe 11 μ Μ, as shown in Figure 5.

EXPERIMENTAL EXAMPLE 8:FP technology proof chemical compound can with the substrate competition binding site.

Fluorescent labeling FTO substrate DNA (5 '-ATTGTCA (6meA) CAGCAGA-3 '-FAM), after fluorescently-labeled DNA and protein binding, effectively molecular change is big, and polarized fluorescence strengthens.Experimental result shows that along with compound concentration increases, polarized fluorescence intensity weakens gradually, and three groups of repetitions are established in experiment, and are as shown in Figure 8.Chemical compound is competed binding site with DNA probably, causes DNA and FTO to dissociate, and fluorescent value reduces gradually.Chemical compound 1 relative other chemical compound has stronger competitiveness, and this is consistent with the active result of inhibition.As shown in Figure 6.

EXPERIMENTAL EXAMPLE 9: chemical compound 1 is in the last inhibitory action to FTO of neuroblastoma cell BE (2)-C.

Research shows that 6meA concentration significantly raises in the HeLa cell RNA that FTO siRNA handles, and FTO crosses expression and then significantly reduces 6meA concentration.Here use 150 μ M and 300 μ M chemical compounds, 1 treatments B E (2)-C cell respectively, HPLC detects the ratio of 6meA/U, 6meA/C, 6meA/G among the RNA, and 6meA concentration significantly raises in the cell, like Fig. 7.

The result shows that chemical compound 1 can be through suppressing the active interior 6meA concentration of cell that improves of FTO.

Claims (3)

1. chrysophanic acid or rhein compound shown in the following general formula I are the purposes in the medicine of disease of target spot with FTO in the preparation treatment:

Wherein, R ₂~ R ₃, R ₆~ R ₇In have at least one for-COOH ,-COONa ,-COOK ,-SO ₃H ,-SO ₃Na ,-SO ₃K; R ₁～R ₈All the other positions be following substituent group :-H ,-OH ,-NH ₂, halogen.

2. purposes according to claim 1, wherein, in the chemical compound shown in the general formula I, R ₁～R ₈For being selected from the substituent group in the following table:

3. purposes according to claim 2, wherein, said compound of Formula I is chrysophanic acid or its sodium salt or potassium salt.

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