CN104316682A - Activity detection method for S-adenosylmethionine decarboxylase (SAMDC) and application thereof - Google Patents
Activity detection method for S-adenosylmethionine decarboxylase (SAMDC) and application thereof Download PDFInfo
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- CN104316682A CN104316682A CN201410530672.3A CN201410530672A CN104316682A CN 104316682 A CN104316682 A CN 104316682A CN 201410530672 A CN201410530672 A CN 201410530672A CN 104316682 A CN104316682 A CN 104316682A
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- adenosylmethionine
- adometdc
- adenosylmethionine decarboxylase
- decarboxylase
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- 108010070753 Adenosylmethionine decarboxylase Proteins 0.000 title claims abstract description 90
- 102000005758 Adenosylmethionine decarboxylase Human genes 0.000 title claims abstract description 90
- 230000000694 effects Effects 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 11
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 claims abstract description 39
- 239000003112 inhibitor Substances 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 20
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- 238000010998 test method Methods 0.000 claims description 11
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 8
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- IDWRPPGWYFFFJY-UHFFFAOYSA-N 3,3-dichloro-6,8-dinitro-1h-quinoline-2,4-dione Chemical compound O=C1C(Cl)(Cl)C(=O)NC2=C1C=C([N+](=O)[O-])C=C2[N+]([O-])=O IDWRPPGWYFFFJY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- CYPGNVSXMAUSJY-CXUHLZMHSA-N (1e)-1-(diaminomethylidenehydrazinylidene)-2,3-dihydroindene-4-carboximidamide Chemical compound C1=CC=C(C(N)=N)C2=C1C(=N/N=C(N)N)/CC2 CYPGNVSXMAUSJY-CXUHLZMHSA-N 0.000 description 1
- PIKMGYXXXIPBPZ-UHFFFAOYSA-N Cc1c(C)[s]c(NC(C[n](cn2)nc2[N+]([O-])=O)=O)c1C(N)=O Chemical compound Cc1c(C)[s]c(NC(C[n](cn2)nc2[N+]([O-])=O)=O)c1C(N)=O PIKMGYXXXIPBPZ-UHFFFAOYSA-N 0.000 description 1
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- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Abstract
The invention relates to an activity detection method for S-adenosylmethionine decarboxylase (SAMDC). The method comprises the following steps: mixing a carbon dioxide determination reagent R1 mixed reagent and R2 mixed reagent in a 1.5mL EP test tube, then adding adenosylmethionine, finally adding SAMDC to obtain a mixed solution with total volume of 1000 microliters, and starting the reaction; performing the incubation reaction on the mixed solution for 1 to 30 minutes in a water bath with the temperature of 37 DEG C, then transferring the mixed solution to a 200 microliters/pore transparent 96-pore plate, detecting the light absorption of the system at the 340nm wavelength by utilizing instruments such as a microplate reader, and detecting the reaction activity of the mixture. The method is simple in operation and good in effect, and the activity of the SAMDC can be detected only by simple light absorption detection. The method is suitable for rapidly detecting the activity of AMDC and screening and designing an inhibitor.
Description
Technical field
The invention belongs to biomedical sector, relate to S adenosylmethionine decarboxylase (S-adenosylmethionine decarboxylase; AdoMetDC), people source S adenosylmethionine decarboxylase activity test method and and application in drug screening is specifically related to.
Background technology
Protein is one of main constituent of biosome, has been the main matter of various vital movement.In various protein, proteinase is most important to vital movement, and the biochemical reaction process in nearly all biosome all carries out catalysis by proteinase.In biosome, the activity of various proteinase has strict regulatory mechanism, once its regulatory mechanism goes wrong, causes the hyperactivity of proteinase, too low or complete deactivation, all can cause corresponding various diseases.Therefore, carried out the activity of modulin enzyme by medicine, make it recover and remain on normal level, there is very important theory significance and realistic meaning.Drug design based on structure is the very important means designing with protein the medicine being target.
Polyamines (polyamines) is the cation micro molecule of the positively charged that a class produces from amino acid metabolism, all exists in all biosomes, and cell growth, differentiation, survival and natural biological function etc. are all indispensable.The characteristic of the many positive charges of polyamines band, make them by forming electrostatic interaction with electronegative biomacromolecule (DNA, RNA, protein, cell membrane etc.), thus regulation and control biological process widely, comprise that chromosome knob is configured to, DNA synthesis with stable, DNA replication dna, transcribe and translate, protein phosphorylation, ribosomes generate, the regulation and control, radicals scavenging etc. of ion channel and film surface receptor.Natural polyamines has a variety of.In mammal, naturally occurring have three kinds, i.e. putrescine (putrescine), spermidine (spermidine), spermine. (spermine), and they are essential with growth to mammal normal growth.Because polyamines has important biological function, its Intracellular levels is subject to strict regulation and control.In the cell (as tumour cell) of Fast-propagation, polyamine level also can rise and lack of proper care.Polyamine level raises, and reduces and expression rising etc. that is tumor-infiltrated and metastasis related gene along with cell proliferation quickening, apoptosis.Therefore, the regulation and control of polyamines, become an important means in oncotherapy and medicament research and development.
AdoMetDC is a key enzyme of synthesis in polyamines body, and the synthesis for spermine and spermidine is most important, because the aminopropyl of spermine and spermidine derives from the decarboxylic reaction of AdoMetDC.Therefore, synthesis AdoMetDC inhibitor, suppresses the generation of spermine and spermidine, is a very concerned at present oncotherapy approach.Meanwhile, because pathogenic microorganism also needs to maintain normal polyamine level, therefore AdoMetDC inhibitor also becomes an important drugs target for pathogenic microorganism.
Current, the inhibitor MGBG (mitoguazone) of AdoMetDC be used to clinical in anti-leukocythemia treatment and the chemotherapy ancillary drug of cancer.But MGBG can cause serious injury of mitochondria, and therefore toxic and side effect is very large.The inhibitor C GP 48664 (SAM486A) of AdoMetDC has entered I phase and II phase clinical assessment.Therefore, screen and design new AdoMetDC inhibitor significant and be worth.
In the process of screening and design AdoMetDC inhibitor, a very important evaluation index is the inhibition of enzyme activity ability of detection of drugs to AdoMetDC.At present, detect the activity of AdoMetDC and inhibitor effect, more effectively adopt the isotope-labeled adenosylmethionine of C14 ([
14cOOH] AdoMet) test, by detecting
14cO
2generation evaluate.Although the method is very sensitive and accurate, owing to relating to isotope operation, need special equipment and operation, also costly, therefore common lab is difficult to use drug price, and cannot be used for high-throughout AdoMetDC drug screening.Therefore, develop the AdoMetDC Activity determination system that cheap, easy and simple to handle, pervasive degree is high, there is important value.
Summary of the invention
The object of the invention is to determine to set up the method for an AdoMetDC Activity determination that is easy, non radioactive isotope mark, for Activity determination and inhibitor evaluation, screening and the design of AdoMetDC.
Technical scheme of the present invention is, produces CO based on decarboxylase
2principle, utilizing the PEPC enzyme process of CO 2 measuring, by regulating relevant constituent, determining a measuring method for activity that can carry out AdoMetDC Activity determination and inhibitor effect and evaluate.R1 and the R2 reagent of the method mainly composed as follows:
The concentration of described adenosylmethionine is 0.01-500mM, and the concentration of S adenosylmethionine decarboxylase (AdoMetDC) is 0.1-100uM.
More preferably:
The mix reagent of described R1 is the mixed solution of the phosphoenolpyruvic acid of 7.0mmol/L and the magnesium chloride of 8.0mmol/L; The mix reagent of R2 is the malic dehydrogenase of the phosphoric acid enol pyruvic acid carboxylase of 400U/L, 600U/L, the mixed solution of the nicotinamide adenine dinucleotide of 0.45mmol/L; The concentration of adenosylmethionine is 1mM; The concentration of S adenosylmethionine decarboxylase (AdoMetDC) is 1uM.
For detecting the activity of AdoMetDC, the chemical reaction that we utilize is, adenosylmethionine is under the catalysis of AdoMetDC, and decarboxylation generates carbon dioxide, and the carbon dioxide produced in aqueous is water-soluble, exists with bicarbonate form.Then PEP and bicarbonate radical are under the catalysis of PEPC, and oxalic salt (Oxalate), NADH under the catalysis of MDH, is reduced to NAD by oxalates further
+.NADH has light absorption at 340nm place, and NAD
+no, therefore can with the reduction of 340nm place system light absorption value, due to CO that decarboxylase catalyzes reaction produces in reflection system
2the change of amount, thus reflect its enzymatic activity.
Utilize above-mentioned CO 2 measuring principle, detect the amount of the carbon dioxide that above-mentioned reaction produces.
Owing to there being carbon dioxide to exist in air, so cover tightly reaction vessel in an experiment as far as possible, avoid the contact with air, and ensure to test in the reaction vessel of inflated with nitrogen.
Concrete operation method is, with the EP test tube of 1.5mL, R1, R2 of CO 2 measuring reagent is mixed, then adds adenosylmethionine, finally adds AdoMetDC proteinase, makes cumulative volume be the mixed liquor of 1000ul, starts reaction.After this mixed liquor is carried out incubation reaction 1-30min in 37 DEG C of water-baths, solution is gone to transparent 96 orifice plates (200uL/ hole), utilize the light absorption of the instrument detection system such as microplate reader at 340nm wavelength place.By not adding the control systems of AdoMetDC or adenosylmethionine, reactivity is judged.
The volume ratio of described R1 mix reagent, R2 mix reagent, adenosylmethionine, S adenosylmethionine decarboxylase (AdoMetDC) is 1:2.8-3.5:0.001-0.003:0.5-0.8.
The method is simple to operate, respond well, only needs simple light absorption to detect, can realize.Therefore, the method is applicable to carry out fast the detection of AdoMetDC activity and the screening of inhibitor and design.
Accompanying drawing explanation
Fig. 1, for not add AdoMetDC, does not add adenosylmethionine (SAM), adds the absorbance of AdoMetDC, adenosylmethionine (SAM).
Fig. 2 is that variable concentrations adenosylmethionine (SAM) is to the effect diagram of reaction system.
Fig. 3 is the inhibition figure that known AdoMetDC inhibitor MGBG utilizes the method to detect.
Fig. 4 is the inhibition figure that AdoMetDC inhibitors 4-(2-{ [(5-phenyl-2-furyl) methyl] amino}ethyl) benzenesulfonamide utilizes the method to detect.
Fig. 5 is AdoMetDC inhibitor
2-[(3-nitro-1H-1,2,4-triazol-1-yl}acetyl) and amino]-4, the 5-dimethyl-3-thiophenecarboxamide inhibition figure utilizing the method to detect.
Fig. 6 is the inhibition figure that AdoMetDC inhibitor 3,3-dichloro-6,8-bisnitro-2,4 (1H, 3H)-quinolinedione utilizes the method to detect.
Embodiment
Embodiment 1
With the EP test tube of 1.5mL, R1715uL, R2235uL of CO 2 measuring reagent are mixed, then adding 2uL concentration is the adenosylmethionine of 500mM, and finally adding AdoMetDC proteinase final concentration is 1uM, make cumulative volume be the mixed liquor of 1000ul, start reaction.Carry out incubation reaction by this mixed liquor 37 C water bath, after 5min, solution is gone to transparent 96 orifice plates (200uL/ hole), utilize the light absorption of the instrument detection system such as microplate reader at 340nm wavelength place.By not adding the control systems of AdoMetDC or adenosylmethionine, reactivity is detected.
Represent in Fig. 1 and do not add AdoMetDC, add adenosylmethionine (SAM), AdoMetDC, do not add the absorbance that adenosylmethionine (SAM) all adds.As can be seen from the figure when generation carbon dioxide is carried out in reaction, system NADH reduces, and light absorption value declines.This result shows, uses the method, effectively can detect the CO that AdoMetDC catalysis SAM produces
2consume the light absorption value decline that NADH causes.
Embodiment 2
With the EP test tube of 1.5mL, R1715uL, R2235uL of CO 2 measuring reagent are mixed, then add respectively 2uL adenosylmethionine (in this experiment design concentration be 0.1,0.25,0.5,1.0,2.5,5.0, a 10mM7 index), finally adding AdoMetDC proteinase final concentration is 1uM, make cumulative volume be the mixed liquor of 1000ul, start reaction.Carry out incubation reaction by this mixed liquor 37 C water bath, after 30min, solution is gone to transparent 96 orifice plates (200uL/ hole), utilize the light absorption of the instrument detection system such as microplate reader at 340nm wavelength place.By not adding the control systems of AdoMetDC or adenosylmethionine, reactivity is detected.
In the method, the concentration of adenosylmethionine is very crucial to the effect of body series, found through experiments, and the concentration of adenosylmethionine is controlled, at 1mM and following, effectively to avoid the impact on this detection system.Fig. 2 is that variable concentrations adenosylmethionine (SAM) is to the effect diagram of reaction system.This figure shows, along with the rising of SAM concentration, can cause the decline of system light absorption value, produces false positive.Do not producing under obvious false-positive prerequisite, the suitableeest working concentration of SAM is 1.0mM.
When AdoMetDC inhibitor screening and effect detection, concrete operation method is similar to the above, but in interpolation AdoMetDC this link, first certain density medicine and AdoMetDC is mixed to be incorporated in 37 DEG C and to hatch 30min.
Embodiment 3
The inhibition figure (as shown in Figure 3) that known AdoMetDC inhibitor MGBG utilizes the method to detect.MGBG is dissolved in DMSO, therefore contrasts with DMSO.Concentration is lower, more weak to the rejection ability of AdoMetDC, therefore light absorption value lower (NADH consumes more).This figure shows, uses the method, effectively can detect the inhibiting effect that known inhibitor MGBG reacts AdoMetDC catalysis SAM, and demonstrate obvious concentration dependant.Before replacing it with figure below.
Embodiment 4
According to above scheme, we utilize AutoDockVina software, by being docked to by the Small molecular in SPECS Small molecular database in AdoMetDC pocket that we determine, the marking provided by AutoDockVina software is evaluated, and minimum marking is used for experimental verification lower than the Small molecular of-7.0.
Involved micromolecular inhibitor is:
4-(2-{ [(5-phenyl-2-furyl) methyl] amino}ethyl) benzenesulfonamide (4-(2-{ [(5-phenyl-2-furyl) methyl] is amino } ethyl) benzsulfamide) there is the active inhibition of obvious AdoMetDC, result is as shown in Figure 4.Small molecular DMSO dissolves, and therefore contrasts with DMSO.AdoMetDC activity is high, then NADH reduces, and light absorption declines.Small molecular suppresses AdoMetDC active, then make light absorption decline and reduce.This figure shows, this activity test method, detects that this inhibitor has obvious suppression when variable concentrations to the activity of AdoMetDC catalysis SAM.Under higher concentration, this inhibitor has better inhibition.
The structural formula of this inhibitor is:
Embodiment 5
According to above scheme, we utilize AutoDockVina software, by being docked to by the Small molecular in SPECS Small molecular database in AdoMetDC pocket that we determine, the marking provided by AutoDockVina software is evaluated, and minimum marking is used for experimental verification lower than the Small molecular of-7.0.
Involved micromolecular inhibitor is:
2-[({ 3-nitro-1H-1; 2; 4-triazol-1-yl}acetyl) amino]-4; 5-dimethyl-3-thiophenecarb oxamide (2-[({ 3-nitro-1H-1; 2; 4-triazol-1-yl } acetyl group) amino]-4,5-dimethyl-3-thenoyl amines) there is the active inhibition of obvious AdoMetDC, result is as shown in Figure 5.Small molecular DMSO dissolves, and therefore contrasts with DMSO.AdoMetDC activity is high, then NADH reduces, and light absorption declines.Small molecular suppresses AdoMetDC active, then make light absorption decline and reduce.Concrete Activity determination step is shown in described in embodiment 1.This figure shows, this activity test method, detects that this inhibitor has obvious suppression when variable concentrations to the activity of AdoMetDC catalysis SAM.Under higher concentration, this inhibitor has better inhibition.
The structural formula of this inhibitor is:
Embodiment 6
According to above scheme, we utilize AutoDockVina software, by being docked to by the Small molecular in SPECS Small molecular database in AdoMetDC pocket that we determine, the marking provided by AutoDockVina software is evaluated, and minimum marking is used for experimental verification lower than the Small molecular of-7.0.
Involved micromolecular inhibitor 3,3-dichloro-6,8-bisnitro-2,4 (1H, 3H)-quinolinedione (chloro-6,8-dinitro-2,4 (1H, the 3H)-quinolizines of 3,3-bis-),
Have the active inhibition of obvious AdoMetDC, result as shown in Figure 6.Small molecular DMSO dissolves, and therefore contrasts with DMSO.AdoMetDC activity is high, then NADH reduces, and light absorption declines.Small molecular suppresses AdoMetDC active, then make light absorption decline and reduce.Concrete Activity determination step is shown in described in embodiment 1.This figure shows, this activity test method, detects that this inhibitor has obvious suppression when variable concentrations to the activity of AdoMetDC catalysis SAM.Under higher concentration, this inhibitor has better inhibition.
The structural formula of this inhibitor is:
Experimental subjects of the present invention is people source AdoMetDc, but have homology between the AdoMetDC due to separate sources, the inhibitor therefore in content of the present invention may can act on the AdoMetDC in other inhuman source or the albumen containing AdoMetDC functional domain equally.
Claims (6)
1. an activity test method for S adenosylmethionine decarboxylase, is characterized in that, comprises the steps:
1) CO 2 measuring reagent R1 mix reagent, R2 mix reagent are mixed at the EP test tube of 1.5mL, add adenosylmethionine again, finally add S adenosylmethionine decarboxylase (AdoMetDC), obtain the mixed liquor that cumulative volume is 1000ul, start reaction;
2) by above-mentioned mixed liquor in the water-bath of 37 DEG C after incubation reaction 1-30min, again this mixed liquor is gone to transparent 96 orifice plates in 200uL/ hole, utilize the light absorption of the instrument detection system such as microplate reader at 340nm wavelength place, the reactivity of this potpourri is detected;
3) in step 1), 2) basis on detect the reactivity of the mixed liquor not adding S adenosylmethionine decarboxylase (AdoMetDC) or do not add adenosylmethionine simultaneously.
2. the activity test method of S adenosylmethionine decarboxylase according to claim 1, is characterized in that: the mix reagent of described R1 is the mixed solution of the phosphoenolpyruvic acid of 5.5-10.5mmol/L and the magnesium chloride of 4.6-10.8mmol/L; The mix reagent of R2 is the phosphoric acid enol pyruvic acid carboxylase of 360-450U/L, the malic dehydrogenase of 520-680U/L, the mixed solution of the nicotinamide adenine dinucleotide of 0.35-0.55mmol/L, the concentration of described adenosylmethionine is 0.01-500mM; The concentration of described S adenosylmethionine decarboxylase (AdoMetDC) is 0.1-100uM.
3. the activity test method of S adenosylmethionine decarboxylase according to claim 1, is characterized in that: the mix reagent of described R1 is the mixed solution of the phosphoenolpyruvic acid of 7.0mmol/L and the magnesium chloride of 8.0mmol/L; The mix reagent of R2 is the malic dehydrogenase of the phosphoric acid enol pyruvic acid carboxylase of 400U/L, 600U/L, the mixed solution of the nicotinamide adenine dinucleotide of 0.45mmol/L; The concentration of adenosylmethionine is 1mM; The concentration of S adenosylmethionine decarboxylase (AdoMetDC) is 1uM.
4. the activity test method of S adenosylmethionine decarboxylase according to claim 1, is characterized in that: the volume ratio of R1 mix reagent, R2 mix reagent, adenosylmethionine, S adenosylmethionine decarboxylase (AdoMetDC) is 1:2.8-3.5:0.001-0.003:0.5-0.8.
5. the activity test method of S adenosylmethionine decarboxylase according to claim 1, is characterized in that: step 1) described in reaction be under air conditions or at N
2carry out under protective condition.
The activity test method of 6.S-adenosylmethionone decarboxylase suppresses the application on the inhibitor of S adenosylmethionine decarboxylase in screening.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320167A (en) * | 1998-09-29 | 2001-10-31 | 新加坡共和国政府 | An i(in vitro) activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening for inhibitors of HBV DNA polymerase |
| CN1690691A (en) * | 2004-04-23 | 2005-11-02 | 中国科学院上海药物研究所 | Process for activity determination of SARS coronavirus 3CL protease and inhibitor screening |
| CN101842008A (en) * | 2007-08-02 | 2010-09-22 | 南方研究所 | 5 '-be substituted adenosine, its preparation and as the purposes of S adenosylmethionine decarboxylase inhibitor |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320167A (en) * | 1998-09-29 | 2001-10-31 | 新加坡共和国政府 | An i(in vitro) activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening for inhibitors of HBV DNA polymerase |
| CN1690691A (en) * | 2004-04-23 | 2005-11-02 | 中国科学院上海药物研究所 | Process for activity determination of SARS coronavirus 3CL protease and inhibitor screening |
| CN101842008A (en) * | 2007-08-02 | 2010-09-22 | 南方研究所 | 5 '-be substituted adenosine, its preparation and as the purposes of S adenosylmethionine decarboxylase inhibitor |
Non-Patent Citations (1)
| Title |
|---|
| 路玉兰等: "百脉根S-腺苷甲硫氨酸脱羧酶基因克隆于表达分析", 《华北农学报》 * |
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