CN101914614A - Method and kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity - Google Patents
Method and kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity Download PDFInfo
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Abstract
The invention discloses a method and a kit for determining nicotinamide phosphoribosyl transferase (Nampt) activity. The method comprises the following steps of: (1) establishing the reaction for converting the Nampt catalytic substrate of nicotinamide (NAM) into the product of nicotinamide mononucleotide (NMN); (2) terminating the reaction by a heating method; (3) adding hypnone and strong base for thorough reaction; (4) adding formic acid for thorough reaction; (5) detecting the fluorescence intensity at the location with the excitation wavelength of 326-426nm and the transmission wavelength of 410-520nm. The invention also provides a method and a system for high-flux sieving of a Nampt inhibitor or activator. The method comprises the following steps of: incubating the compound candidate and the reacting system for the Nampt (except the substrate of NAM) in a micropore plate; determining the Nampt activity in accordance with the steps. The method has the advantages of simple operation, moderate reaction condition and high-flux operation.
Description
Technical field
The invention belongs to chemical field, particularly a kind of method and test kit thereof of measuring the Nampt enzymic activity.The present invention also provides the high-throughput screening method and the screening system thereof of a kind of Nampt enzyme inhibitors or activator.
Background technology
Niacinamide phosphoribosyltransferase (Nampt has another name called plain visfatin of interior fat or pre B cell clone enhancement factor PBEF) is an albumen indispensable in the Mammals vital movement.It is early stage that the homozygote mouse of Nampt defective (Nampt-/-) dies from fetal development.The level of essential energy matter NAD coenzyme in its regulation and control mammalian cell, have angiogenic growth, anti-apoptotic, participation body inflammatory and reply, promote various kinds of cell differentiation and maturation, potential to activate physiological functions such as progenitor cell, have the potential effect for diseases prevention and treatment such as cancer, cerebral apoplexies.Tumour cell has very high NAD and consumes and metabolic rate, and the rate-limiting enzyme Nampt of NAD route of synthesis is the target of prevention and control of cancer, its enzyme inhibitors FK866 (Hasmann M, et al., Cancer Research 2003; 63:7436-7442.) and CHS-828 (HjarnaaPJV, et al., Cancer Research 1999; 59:5751-5757.) the present preclinical study of cancer therapy that all entered.On the other hand, NAD has neuroprotective (Liu D, et al., Neuromolecular Medicine 2009; 11:28-42), and Nampt can prevent and treat brain injury (the Zhang W that cerebral apoplexy causes, et al., J Cereb Blood Flow Metab.2010May 19.Epub ahead of print), can be used as the new target drone of cerebral apoplexy control, raise the Nampt enzymic activity and will help tackling the multiple pathological factor that development takes place cerebral apoplexy, so the Nampt activator may become the new drug of effective control cerebral apoplexy.
Cancer, cerebral apoplexy are the two big important diseases that threaten human health and life, also are the focuses that global life science and medical field are paid close attention to, and find that therefore the small molecules of regulating the Nampt enzymic activity will have great importance to the control of these major diseases.High flux screening is one of important means of new drug development, and the screening system of molecular level helps to improve breakneck acceleration, increases the screening flux, reduces the screening cost.Yet also be not the screening system report of target at present with Nampt; The Nampt activity inhibitor has only been reported 3, FK866, CHS-828 and based on molecule I S001 (Kang GB, et al., the Molecules and Cells 2009 of FK866 structure of modification; 27:667-671.); And Nampt zymoexciter none example report still so far.
The Nampt activity determination method of having reported at present mainly contains three kinds: use radioactive substrates to obtain radiolabeled product; Use the HPLC reaction product isolated; Is NAD with product NMN through the Nmnat enzymatic conversion, is converted into fluorescence-causing substance NADH by ethanol dehydrogenase again.These methods or operate dangerous, perhaps complex steps, perhaps cost is higher, all is not suitable for the flux operation.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly to realize the defective of high-throughput operation at the method for existing mensuration Nampt enzymic activity, a kind of method and test kit thereof of the Nampt of mensuration enzymic activity are provided, and this method is simple to operate, can realize the high-throughput operation.The present invention also provides the high-throughput screening method and the screening system thereof of a kind of Nampt enzyme inhibitors or activator.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: the active method of a kind of mensuration niacinamide phosphoribosyltransferase may further comprise the steps:
(1) sets up the reaction that Nampt substrate for enzymatic activity niacinamide (NAM) changes into product nmn (NMN);
(2) heating method stops enzyme reaction;
(3) adding methyl phenyl ketone and highly basic fully reacts;
(4) adding formic acid more fully reacts; With
(5) detect excitation wavelength 326～426nm, the fluorescence intensity at emission wavelength 410～520nm place.
Wherein, in the step (1), the reaction system of described reaction can be that the conventional Nampt enzyme catalysis NAM that is applicable to changes into the reaction system of NMN, and preferable comprises: niacinamide phosphoribosyltransferase, substrate NAM, ATP, PRPP, damping fluid, MgCl
2, BSA, DTT and DMSO.Wherein, Nampt enzyme reaction concentration is 1～8 μ g/ml preferably, more preferably 2 μ g/ml.Substrate NAM reaction density is 0.2～2 μ M preferably, more preferably 0.2 μ M.DMSO concentration preferably 1～8%, more preferably 2%.The component that comprises following each concentration that the reaction system of described reaction is better: 50mMTris-HCl (pH7.5), 0.02%BSA, 12mM MgCl
2, 2mM ATP, 0.4mM PRPP, 2mMDTT, 2 μ g/ml Nampt, 0.2 μ M NAM and 2%DMSO.The reaction conditions of described reaction is the reaction conditions of the Nampt enzyme of routine.The optimal reaction temperature of Nampt enzyme is 37 ℃.Preferably 10～60 minutes enzyme reaction time, more preferably 10～30 minutes, best was 15 minutes.
In the step (2), described heating method is the method for the termination enzyme reaction of routine, and heating makes enzyme denaturation and loses activity, thereby enzyme reaction stops.Preferably 95 ℃ were heated 1 minute.
In the step (3), preferably 0～25 ℃ of the temperature of reaction of described reaction, more preferably 0 ℃, preferably 5～30 minutes reaction times, more preferably 5～10 minutes.Methyl phenyl ketone and alkaline reaction density are preferable is respectively 0.222M, 2.2%.Among the present invention, described highly basic is NaOH or KOH preferably, more preferably KOH.
In the step (4), what described reaction was preferable is 70～90 ℃ of reactions 5～15 minutes, and more preferably 70 ℃ were reacted 5 minutes.
In the step (5), the detection method of described fluorescence intensity is a prior art.Preferable use microplate reader detects.The wavelength region of fluoroscopic examination: excitation wavelength 326～426nm, emission wavelength 410～520nm, maximum excitation wavelength are 382nm, the optimum transmit wavelength is 445nm.
The present invention also provides a kind of mensuration niacinamide phosphoribosyltransferase active test kit, comprise: niacinamide phosphoribosyltransferase (Nampt) and reaction substrate, described reaction substrate is NAM (niacinamide) and PRPP (ribose 5-phosphate-1-tetra-sodium), also comprises methyl phenyl ketone, highly basic and formic acid.
Among the present invention, described test kit is preferable also further comprises reaction buffer.Described reaction buffer is the damping fluid that Nampt substrate for enzymatic activity niacinamide (NAM) and PRPP generation product nmn (NMN) are carried out in conventional being used to.The concentrated solution that preferably comprises following component solution: 50mM Tris-HCl (pH7.5), 0.02%BSA, 12mM MgCl
2, 2mM ATP, 2mM DTT and 2%DMSO.Each component is preferable working concentration in the solution of above-mentioned concentration, and each component concentrations is its multiple in the described concentrated solution, and this cycles of concentration can be conventional, preferably 10 times.DMSO concentration preferably 1～8%, more preferably 2%.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: the high-throughput screening method of a kind of niacinamide phosphoribosyltransferase inhibitor or activator may further comprise the steps:
1. in microwell plate, add candidate compound and the niacinamide phosphoribosyltransferase reaction system except that substrate NAM, hatch;
2. add substrate NAM and start reaction;
3. heating method stops enzyme reaction;
4. add methyl phenyl ketone and highly basic fully reacts;
5. adding formic acid more fully reacts; With
6. detect excitation wavelength 326～426nm, the fluorescence intensity at emission wavelength 410～520nm place.
Among the present invention, set up following control reaction in the time of preferable:
Do not contain substrate and compound and carry out enzyme reaction and detection reaction, the fluorescent value that records is defined as system background fluorescence value F
Bg
Add substrate, do not add compound and carry out enzyme reaction and detection reaction, the baseline fluorescence values F of system when the fluorescent value that records is defined as no compound intervention
Ref
Add substrate simultaneously, compound carries out enzyme reaction and detection reaction, the fluorescent value that records is the fluorescent value F of system under the compound N intervention
N
In the method for the present invention, the enzymic activity that setting system background response records is 0%, and the enzymic activity that the benchmark reaction records is 100%, can try to achieve the enzymic activity E of compound according to formula (1)
N:
E
N＝(F
N-F
bg)/(F
ref-F
bg)×100％ (1)。
Screening method of the present invention, the preferable step of the candidate's active compound that screens being carried out multiple sieve that also comprises: repeat above-mentioned screening step when sieving again 1.～6., preferable three groups of control reaction that in above-mentioned screening process, relate to, set up one group of control reaction again: add compound and do not add substrate and carry out successively measuring fluorescent value (F after enzyme reaction and the detection reaction to get rid of false positive results
C-bg).
One preferred embodiment of the high-throughput screening method of niacinamide phosphoribosyltransferase inhibitor of the present invention or activator is may further comprise the steps:
(1) enzyme reaction:
The enzyme reaction system is 25 μ l, comprises the component of following each concentration: 50mM Tris-HCl (pH7.5), 0.02%BSA, 12mM MgCl
2, 2mM ATP, 0.4mM PRPP, 2mM DTT, 2 μ g/mlNampt, 0.2 μ M NAM, 20 μ M candidate compound and 2%DMSO;
DMSO solution with 0.5 μ l compound is added on 96 orifice plates earlier, add 20 μ l enzyme reaction mixing solutionss (the enzyme reaction component except that substrate NAM) again, behind 37 ℃ of preincubate 5min, add 4.5 μ l substrate NAM solution to start reaction, stop enzyme reaction in 95 ℃ of heating 1min behind 37 ℃ of reaction 15min;
(2) detection reaction:
1. treat enzyme reaction solution after cooled on ice, add 10 μ l, 20% methyl phenyl ketone and 2M KOH successively, on the vortex mixed instrument behind the mixing in 0 ℃ the effect 10min, add 45 μ l, 88% formic acid, 70 ℃ the heating 5min, cooled on ice;
2. use microplate reader to measure the fluorescence intensity at excitation wavelength 382nm, emission wavelength 445nm place.
The present invention also provides the high throughput screening system of a kind of niacinamide phosphoribosyltransferase inhibitor or activator, comprising: microwell plate;
Niacinamide phosphoribosyltransferase (Nampt) and reaction substrate, described reaction substrate are NAM (niacinamide) and PRPP (ribose 5-phosphate-1-tetra-sodium);
Methyl phenyl ketone, highly basic and formic acid.
Wherein, described microwell plate is as indicated above can be conventional experiment consumptive material, as 48 orifice plates, 96 orifice plates, 384 orifice plates etc.Described screening system is preferable also comprises reaction buffer.Described reaction buffer is as indicated above, is the damping fluid that Nampt substrate for enzymatic activity niacinamide (NAM) and PRPP generation product nmn (NMN) are carried out in conventional being used to.The concentrated solution that preferably comprises following component solution: 50mM Tris-HCl (pH7.5), 0.02%BSA, 12mM MgCl
2, 2mM ATP, 2mM DTT and 2%DMSO.Each component is preferable working concentration in the solution of above-mentioned concentration, and each component concentrations is its multiple in the described concentrated solution, and this cycles of concentration can be conventional, preferably 10 times.DMSO concentration preferably 1～8%, more preferably 2%.Described screening system is preferable also further comprises positive control and negative control.Described positive control is known niacinamide phosphoribosyltransferase inhibitor or activator, as FK866.Described negative control is the solvent of dissolving candidate compound.Described screening system is preferable also comprises microplate reader.
Among the present invention, but above-mentioned optimum condition arbitrary combination on the basis that meets this area general knowledge promptly gets the preferred embodiments of the invention.
" room temperature " described in the present invention is meant the temperature of the operation room of testing, and is generally 25 ℃.
The present invention is except that specifying, used per-cent all is mass percent.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art, beneficial effect of the present invention is as follows: the method that the present invention sets up need not separated by detecting the fluorescent signal of end product, and is simple to operate; Do not use the medicine of radioactivity or severe toxicity, the reaction conditions gentleness, it is cheap to detect safety; Enzyme reaction is adapted on 96 orifice plates or the enterprising quantized operation that works of microwell plate such as 384 orifice plates.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
The reaction principle that Fig. 1 .Nampt enzymic activity detects.
The fluorescence excitation spectrum and the emmission spectrum of Fig. 2 .NMN derivative.
Fig. 3. detection reaction temperature of reaction and influence of time in second step.
Fig. 4. the influence in reaction times in the detection reaction the first step.
Fig. 5. the relation of fluorescent signal and enzyme product NMN.
The detection of Fig. 6 .Nampt enzymic activity.
Fig. 7. the checking of enzyme reaction terminating method.
Fig. 8. the mensuration of milosevic constant km value.
Fig. 9. the kinetics of enzyme reaction.
Figure 10 .DMSO is to the influence of fluorescence-causing substance.
Figure 11. the preincubate time is to the influence of fluorescence-causing substance.
Figure 12. the primary dcreening operation result of compound.
Figure 13. the result that compound N RC0553 sieves again.
Figure 14. the IC50 pH-value determination pH of compound F 17-hydroxy-corticosterone K866 and Weibull.
Embodiment
Embodiments of the invention are to specify of the present invention, but the present invention is not limited.Not marked actual conditions in the following content of the present invention, usually according to normal condition, or the condition of advising according to manufacturer.
The foundation and the optimization of embodiment 1 enzymic activity detection method
The inventor is successful expression, purifying Nampt recombinant protein from protokaryon intestinal bacteria system, and has set up Nampt enzymic activity detection method, reaction principle as shown in Figure 1, the Nampt enzyme is converted into product nmn (NMN) with substrate niacinamide (NAM); Through two step simple chemical reaction, NMN is converted into the derivative of fluorescence, can detect maximum fluorescence signal (Fig. 2) at excitation wavelength 382nm, emission wavelength 445nm.
The contriver has investigated the influence factor of each step of detection reaction, to obtain the optimum reaction condition in each step.For investigating the influence of detection reaction reaction times and temperature of reaction in second step, in being the NMN of 2 μ M, 25 μ l concentration add 20% methyl phenyl ketone of 10 μ l and 2M KOH successively behind 0 ℃ of reaction 20min, the 88% formic acid mixing that adds 45 μ l again, respectively behind 70 ℃, 80 ℃, 90 ℃ reactions 5min, 10min, 15min in cooled on ice.The detected result of end product fluorescence intensity as shown in Figure 3,70～90 ℃ temperature range and the time span of 5～15min are all less to the influence of fluorescent signal, promptly this step has been finished reaction in the 5min under 70 ℃ temperature condition, and product all keeps stable at higher temperature (to 90 ℃) with in the longer time (to 15min).
Carry out the detection reaction the first step 0 ℃ and room temperature respectively, when finding reaction 15min, the fluorescent signal of gained end product is stronger under 0 ℃ of condition.Therefore under 0 ℃, investigated the influence in reaction times in this step.In 25 μ l concentration is among the NMN of 2 μ M, adds 20% methyl phenyl ketone and the 2M KOH of 10 μ l successively, in 0 ℃ act on 5,10,15,20 respectively, behind the 30min, every pipe adds 88% formic acid of 45 μ l, behind the mixing in 70 ℃ of reaction 5min, cooled on ice.As above survey fluorescent value, the results are shown in Figure 4.As seen from Figure 4, this step has reacted completely when 0 ℃ of following 5min, and in the time range of 5～30min, continue prolonging action time does not have influence to the fluorescent signal of end product.
Therefore, the contriver determines that the detection reaction condition is: 0 ℃ of reaction of the first step 10min, second step, 70 ℃ of reactions 5min.
Then investigated the relation between the fluorescent signal of NMN concentration and product.To carry out detection reaction after the serial dilution of NMN solution, microplate reader is measured the fluorescence intensity of end product.As shown in Figure 5, in 0～80 μ M scope, the fluorescent signal of the concentration of NMN and the derivative of generation is linear at least.Our substrate NAM that experiment showed, does not participate in this chemical reaction simultaneously, can not disturb detecting to produce; And the reaction that does not add enzyme or do not add substrate all can not generate the product that fluorescence be arranged through detection reaction, and and if only if, and substrate for enzymatic activity NAM has generated enzyme product NMN, just can record fluorescent signal (Fig. 6).Therefore this method can be carried out the vitro detection of Nampt enzymic activity by quantitative assay enzyme product.
In order to guarantee the controllability of enzyme reaction time, the contriver has attempted highly basic respectively or the pyritous method stops enzyme reaction.By Fig. 7 A as seen, enzyme reaction can not be carried out in the presence of KOH, but enzyme product NMN also can be degraded by it, and the fluorescence intensity relative comparison group of end product significantly reduces, and therefore is not useable for termination reaction.Nampt enzyme after 95 ℃ of heating 1min sex change is used for enzyme reaction, and the end product fluorescence intensity of detection reaction gained shows that near fluorescence background value (Fig. 7 B) this condition can make the Nampt sex change lose enzymic activity; The reaction solution that to finish simultaneously after the enzyme reaction places 95 ℃ of heating 1min, compares the fluorescent signal (Fig. 7 B) of not remarkably influenced end product with the control group of not handling; Find that in addition the NMN of 95 ℃ of heating 1min processing compares no significant difference through the fluorescent value of gained end product after the detection reaction with undressed NMN, so heat treated can not exert an influence (Fig. 7 C) to the stability of enzyme product NMN yet.To sum up, 95 ℃ of heating 1min can be used for stopping enzyme reaction, and can not influence the stability of follow-up detection reaction or destructive enzyme product NMN.
In enzymatic analysis, Michaelis-Menton constant K
mBe an essential characteristic constant of enzyme, it is comprising enzyme-to-substrate combination and dissociated character.Michaelis-Menton equation is: v=V
Max* [S]/(K
m+ [S]), v is a speed of response in the formula; K
mBe Michaelis-Menton constant; V
MaxBe the enzyme reaction top speed; [S] is concentration of substrate.From Michaelis equation as seen, Michaelis-Menton constant K
mEqual the concentration of substrate that speed of response reaches maximum reaction velocity one half.
The contriver has investigated the relation between substrate NAM concentration and the end product fluorescence intensity.With after the substrate NAM serial dilution respectively with the enzyme of 2 μ g/ml and 37 ℃ of reaction 30min, measure the fluorescence intensity of detection reaction product, as shown in the figure, concentration of substrate is in 0～0.6 μ M scope, reaction product is linear to be increased, and concentration of substrate reaches platform greater than 1.25 μ M afterreactions.Match gets the Michaelis-Menton constant K of Nampt to NAM according to Michaelis-Menton equation
mBe 0.16 μ M (Fig. 8).
In order to save the usage quantity of enzyme, substrate, reach signal to noise ratio preferably simultaneously, the contriver has investigated the influence to reaction product of enzyme and concentration of substrate.In enzyme concn is under the condition of 2,4,8 μ g/ml, and the substrate with 0.2,1,2 μ M concentration carries out enzyme reaction and detection reaction respectively, and microplate reader is measured the fluorescent value of end product, and is as shown in table 1.2 μ g/ml enzyme concns reach capacity to 0.2 μ M concentration of substrate, increase enzyme concn and do not increase the product amount.Though concentration of substrate increases the detection signal that makes end product and increases, and also can increase the consumption of the used compound of screening greatly, therefore is chosen near K
mCarry out enzyme reaction under the concentration of substrate 0.2 μ M of value and the 2 μ g/ml enzyme concns.
The fluorescent value of the reaction of different enzyme concns of table 1. and concentration of substrate
Carry out enzyme reaction according to above-mentioned condition, and stop in differential responses time point heat respectively, enzyme reaction solution is stored in-20 ℃ earlier, treat that all samples finishes to carry out detection reaction simultaneously after the reaction.As seen from Figure 9, be linear increment 30 minutes reaction product with the reaction times, the reaction of substrate and enzyme reaches plateau behind the 45min, and fluorescent derivative no longer increases with the prolongation in reaction times.Reaction is in linear growth and is beneficial to the influence of investigation compound for enzymic activity during the phase, therefore determines that the enzyme reaction time when determination of activity is 15min.
In screening process, for making experimental implementation unitized, all samples all is dissolved among the organic solvent DMSO, therefore investigated the influence of DMSO to the Nampt enzymic activity, as shown in figure 10, with the increase of DMSO concentration (1～8%) in the system, the Nampt enzymic activity is not significant to be changed, and can be chosen in the screening of carrying out compound among the 2%DMSO thus.
Some active compound is slower with combining of enzyme, needs certain hour just can reach balance, for fear of leaking the slow bonded compound of this class of sieve, guarantees the abundant effect of itself and enzyme, need be with compound and enzyme solution preincubate certain hour before reaction.Therefore investigated of the influence of preincubate time to enzymic activity.The DMSO of final concentration 2% and Nampt enzyme buffer liquid at 37 ℃ of preincubate certain hours, are added NAM then and start enzyme reaction.As shown in figure 11, the time of preincubate 0-30min is substantially to not influence of enzymic activity.
The foundation and the quality control of embodiment 3 high flux screening models
3.1 the flow process of high flux screening:
The flow process of high flux screening is as follows:
1, enzyme reaction system is 25 μ l, and wherein various component concentrations are: 50mM Tris-HCl (pH7.5), 0.02%BSA, 12mM MgCl2,2mM ATP, 0.4mM PRPP, 2mM DTT, 2 μ g/mlNampt, 0.2 μ M NAM, 20 μ M candidate compound and 2%DMSO.DMSO solution with 0.5 μ l candidate compound is added on 96 orifice plates earlier, add 20 μ l enzyme reaction mixing solutionss (the enzyme reaction component except that substrate) again, behind 37 ℃ of preincubate 5min, add 4.5 μ l substrate NAM solution to start reaction, stop enzyme reaction in 95 ℃ of heating 1min behind 37 ℃ of reaction 15min.
2, treat enzyme reaction solution after cooled on ice, add 10 μ l, 20% methyl phenyl ketone and 2M KOH successively, on the vortex mixed instrument behind the mixing in 0 ℃ the effect 10min, add 45 μ l, 88% formic acid, 70 ℃ the heating 5min, cooled on ice.
3, use microplate reader to measure the fluorescent value at excitation wavelength 382nm, emission wavelength 445nm place.
3.2 the variation coefficient of high flux screening:
Do not add compound and only add 0.5 μ l DMSO in every hole of microwell plate, according to above-mentioned high flux screening flow process, carry out enzyme reaction and detection reaction successively, last microplate reader is measured fluorescent value (table 2).Add up the variation coefficient of this screening process,, whether can be used for screening in order to assess the requirement whether this screening model satisfies quality-controlling parameters.The variation coefficient claims " standard rate " again, is a statistic weighing the degree of variation of each take off data.The calculation formula of the variation coefficient is: CV=SD/Avg * 100% (SD is that standard deviation, Avg are mean value), it is generally acknowledged that in high flux screening 10% can allow with interior CV value.Statistic data shows that in this application of sample process, the CV value of monoblock 96 orifice plates is 3%, and the CV value of every row and every row is shown in table 3,4, all CV all are lower than 4%, as seen application of sample is accurate in this screening process, reacts controlled, detecting instrument stable reading, meets the requirement of high flux screening.
The fluorescent value of table 2.96 orifice plate screening assay
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | 102 | 109 | 103 | 106 | 104 | 103 | 105 | 103 | 106 | 103 | 105 | 105 |
| B | 103 | 103 | 104 | 103 | 101 | 106 | 103 | 104 | 107 | 103 | 104 | 105 |
| C | 105 | 103 | 105 | 101 | 108 | 103 | 108 | 102 | 107 | 105 | 103 | 104 |
| D | 104 | 109 | 102 | 102 | 103 | 107 | 103 | 106 | 107 | 104 | 107 | 105 |
| E | 104 | 107 | 107 | 101 | 103 | 109 | 105 | 103 | 108 | 103 | 104 | 105 |
| F | 103 | 106 | 106 | 107 | 105 | 106 | 106 | 106 | 107 | 107 | 108 | 102 |
| G | 108 | 101 | 106 | 105 | 103 | 108 | 104 | 105 | 105 | 107 | 103 | 107 |
| H | 106 | 105 | 106 | 108 | 105 | 108 | 102 | 103 | 108 | 106 | 107 | 101 |
The CV value of the every row of table 3.
The CV value of the every row of table 4.
3.3 the Z ' factor of high flux screening:
Z ' the factor (Z ' factor) be an important statistics parameter weighing the high flux screening reliability, it combines the factor of measuring error and signal to noise ratio (signal/noise ratio) two aspects.Z ' factor calculation formula is as follows:
Wherein, SD
MaxFor the survey live body system (the negative live body system that surveys) that does not contain inhibitor surveys and lives, the standard deviation of gained activity between multiple hole; SD
MinFor the survey live body system (the positive live body system that surveys) of the inhibitor that adds high density surveys and lives, the standard deviation of gained activity between multiple hole; Avg.
MaxThe active mean value of negative survey live body system's survey gained alive; Avg.
MinThe active mean value of positive survey live body system's survey gained alive.The negative live body system that surveys claims high control, and the positive live body system that surveys claims low control.Z ' the factor is greater than 0.5, and it is that reliability is higher that live body is surveyed in expression, is fit to carry out high flux screening.
Sample as shown in table 5 is arranged, low control reaction group adds DMSO solution (the positive live body system that surveys of 0.5 μ l, 0.5 μ M FK866, final concentration is 10nM FK866,2%DMSO), High control reaction group adds 0.5 μ l DMSO solution (the negative live body system that surveys, final concentration is 2%DMSO), flow process according to high flux screening is reacted, microplate reader is measured the fluorescence intensity of end product, Z ' the factor of calculating this screening model according to Z ' factor calculation formula is 0.668, greater than 0.5, this result proves that screening system of the present invention is believable.
The measurement result of the table 5.Z ' factor
Carry out the primary dcreening operation and the multiple sieve of compound according to following experimental design.
A. primary dcreening operation
Adopt the flow process of the high flux screening of the foregoing description 3, the DMSO solution of drawing 0.5 μ l sample from the candidate compound motherboard carries out primary dcreening operation under 20 μ M compound concentrations to 96 hole screen plates.Experimental design is as shown in table 6: do not contain substrate and compound and carry out enzyme reaction and detection reaction, the fluorescent value that records is defined as system background fluorescence value Fbg; Add substrate, do not add compound and carry out enzyme reaction and detection reaction, the baseline fluorescence values F of system when the fluorescent value that records is defined as no compound intervention
RefAdd substrate simultaneously, compound carries out enzyme reaction and detection reaction, the fluorescent value that records is the fluorescent value F of system under the compound N intervention
NThe enzymic activity that setting system background response records is 0%, and the enzymic activity that the benchmark reaction records is 100%, can try to achieve the enzymic activity E of compound according to formula (1)
N
E
N＝(F
N-F
bg)/(F
ref-F
bg)×100％ (1)
The experimental design of table 6. primary dcreening operation
B. sieve again
Adopt the flow process of the high flux screening of the foregoing description 3, candidate's active compound of picking out carries out multiple sieve according to the experimental design of table 7.Three group reactions that relate in the primary dcreening operation process, set up three groups of control reaction again to get rid of false positive results: it is the contrast of compound test reaction background that control group 1 is set, and compound is directly carried out measuring fluorescent value (F after the detection reaction
C-bg1), the interference that produces with the fluorescence background of getting rid of compound self; Control group 2 is set for the contrast of compound test reaction background, compound is directly carried out measuring fluorescent value (F after the detection reaction
C-bg2), to get rid of compound is converted into fluorescence-causing substance in detection reaction interference; It is the enzyme reaction contrast that control group 3 is set, and the adding compound does not add substrate to carry out measuring fluorescent value (F after enzyme reaction and the detection reaction successively
C-bg3), may become the substrate of Nampt to get rid of compound, be converted into fluorescence-causing substance after experience enzyme reaction and the detection reaction and the interference that produces.
Table 7. sieves experimental design again
Be feasibility and the reliability of verifying this screening model, 84 micromolecular compounds are carried out primary dcreening operation according to the experimental design of table 6, comprising known Nampt enzyme inhibitors FK866.The result as shown in figure 12, primary dcreening operation has detected this known inhibitor FK866, the compound Weibull of finding the new texture type simultaneously has about 80% inhibiting rate, NCRC0553 has 2.3 times activity ratio.
Above-mentioned 3 candidate's active compounds are carried out multiple sieve, and the result shows that FK866 and Weibull itself do not have fluorescence, and all can not be converted into fluorescence-causing substance in enzyme reaction and detection reaction, does not influence the fluorescent signal of NMN derivative, is effective inhibitor of Nampt enzyme.The multiple sieve result of compound N CRC0553 as shown in figure 13, the fluorescent value F of compound itself
C-bg1With system background fluorescence value F
BgApproaching.Show that compound N CRC0553 itself does not produce fluorescent signal, but significantly strengthen that it is F that compound N CRC0553 adding reaction system records fluorescent value F through its fluorescent signal after the detection reaction
RefAnd F
C-bg2Add and the result, promptly this compound is converted into fluorescent substance in detection reaction, is false positive results.
Further FK866 and Weibull are measured IC
50Value.With DMSO doubling dilution compound solution, and get 0.5 μ l and add successively in the sample well of 96 orifice plates, adopt the flow process of the foregoing description 3, measure the enzymic activity of different concns compound under existing, according to formula (2): E=R/ (1+ (C/IC
50)
S(wherein E is an enzymic activity to)+B, and C is a compound concentration, R, IC
50, S, B be the parameter for the treatment of match), enzymic activity is carried out match to the curve of compound concentration, obtain the IC of FK866 and Weibull
50Value is respectively: 0.9nM and 1.3 μ M (Figure 14).Therefore, use method of the present invention and can find effective enzymic activity regulatory molecule.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's claims institute restricted portion equally.
Claims (10)
1. measure the active method of niacinamide phosphoribosyltransferase for one kind, it is characterized in that, may further comprise the steps:
(1) sets up the reaction that Nampt substrate for enzymatic activity niacinamide (NAM) changes into product nmn (NMN);
(2) heating method stops enzyme reaction;
(3) adding methyl phenyl ketone and highly basic fully reacts;
(4) adding formic acid more fully reacts; With
(5) detect excitation wavelength 326～426nm, the fluorescence intensity at emission wavelength 410～520nm place.
2. the method for claim 1 is characterized in that, the reaction system of reaction comprises described in the step (1): niacinamide phosphoribosyltransferase, substrate NAM, ATP, PRPP, damping fluid, MgCl
2, BSA, DTT and DMSO, the reaction conditions of described reaction is room temperature～37 ℃, 10～60 minutes;
Heating method described in the step (2) is 95 ℃ of heating 1 minute;
Reaction described in the step (3) is 0～25 ℃, and 5～30 minutes, methyl phenyl ketone and alkaline reaction density were respectively 0.222M, 2.2%;
Reaction described in the step (4) is 70～90 ℃ of reactions 5～15 minutes, and the reaction density of formic acid is 44%.
3. one kind is fit to the used active test kit of mensuration niacinamide phosphoribosyltransferase of method as claimed in claim 1 or 2, it is characterized in that, comprise: niacinamide phosphoribosyltransferase (Nampt) and reaction substrate, described reaction substrate is niacinamide (NAM) and ribose 5-phosphate-1-tetra-sodium (PRPP), also comprises methyl phenyl ketone, highly basic and formic acid.
4. test kit as claimed in claim 3 is characterized in that, also comprises reaction buffer, is 50mM Tris-HCl, 0.02%BSA, the 12mMMgCl of the concentrated solution that comprises following component solution: pH7.5
2, 2mMATP, 2mM DTT and 2%DMSO.
5. the high-throughput screening method of niacinamide phosphoribosyltransferase inhibitor or activator is characterized in that, may further comprise the steps:
1. in microwell plate, add candidate compound and the niacinamide phosphoribosyltransferase reaction system except that substrate NAM, hatch;
2. add substrate NAM and start reaction;
3. heating method stops enzyme reaction;
4. add methyl phenyl ketone and highly basic fully reacts;
5. adding formic acid more fully reacts; With
6. detect excitation wavelength 326～426nm, the fluorescence intensity at emission wavelength 410～520nm place.
6. high-throughput screening method as claimed in claim 5 is characterized in that,
Step is hatched described in 1. and is 37 ℃ and hatched 0～30 minute;
Step 2. in, the reaction system of described reaction comprises: niacinamide phosphoribosyltransferase, substrate NAM, ATP, PRPP, damping fluid, MgCl
2, BSA, DTT and DMSO;
The reaction conditions of described reaction is room temperature～37 ℃, 10～60 minutes;
The heating method of step described in 3. is 95 ℃ of heating 1 minute;
The reaction of step described in 4. is 0～25 ℃, and 5～30 minutes, methyl phenyl ketone and alkaline reaction density were respectively 0.222M, 2.2%;
70～90 ℃ of reactions of the reaction of step described in 5. 5～15 minutes, the reaction density of formic acid is 44%;
Step is 6. middle uses microplate reader to detect.
7. high-throughput screening method as claimed in claim 5 is characterized in that, described high-throughput screening method may further comprise the steps:
(1) enzyme reaction:
The enzyme reaction system is 25 μ l, comprises the component of following each concentration: 50mM Tris-HCl (pH7.5), 0.02%BSA, 12mM MgCl
2, 2mM ATP, 0.4mM PRPP, 2mM DTT, 2 μ g/mlNampt, 0.2 μ M NAM, 20 μ M candidate compound and 2%DMSO;
DMSO solution with 0.5 μ l compound is added on 96 orifice plates earlier, add the enzyme reaction mixing solutions of 20 μ l except that substrate NAM again, behind 37 ℃ of preincubate 5min, add 4.5 μ l substrate NAM solution, stop enzyme reaction in 95 ℃ of heating 1min behind 37 ℃ of reaction 15min to start reaction;
(2) detection reaction:
1. in enzyme reaction solution, add 10 μ l, 20% methyl phenyl ketone and 2M KOH successively, in 0 ℃ of effect 10min, add 45 μ l, 88% formic acid behind the mixing, 70 ℃ of heating 5min, cooled on ice;
2. use microplate reader to measure the fluorescence intensity at excitation wavelength 382nm, emission wavelength 445nm place.
8. one kind is fit to the used niacinamide phosphoribosyltransferase inhibitor of method as claimed in claim 5 or the high throughput screening system of activator, it is characterized in that, comprising:
Microwell plate;
Niacinamide phosphoribosyltransferase (Nampt) and reaction substrate, described reaction substrate are niacinamide (NAM) and ribose 5-phosphate-1-tetra-sodium (PRPP);
Methyl phenyl ketone, highly basic and formic acid.
9. high throughput screening system as claimed in claim 8 is characterized in that described screening system also comprises reaction buffer, is the concentrated solution that comprises following component solution: 50mMTris-HCl (pH7.5), 0.02%BSA, 12mM MgCl
2, 2mM ATP, 2mM DTT and 2%DMSO.
10. high throughput screening system as claimed in claim 8 or 9, it is characterized in that, described screening system also further comprises positive control and negative control, described positive control is known niacinamide phosphoribosyltransferase inhibitor or activator, and described negative control is the solvent of dissolving candidate compound.
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| CN103898076A (en) * | 2014-02-24 | 2014-07-02 | 中国科学院武汉物理与数学研究所 | Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor |
| WO2018023206A1 (en) * | 2016-07-30 | 2018-02-08 | 邦泰生物工程(深圳)有限公司 | Nicotinamide phosphoribosyltransferase mutant and application thereof |
| CN107889505A (en) * | 2016-07-30 | 2018-04-06 | 邦泰生物工程(深圳)有限公司 | A kind of method for preparing nicotinamide mononucleotide |
| CN114264822A (en) * | 2020-09-16 | 2022-04-01 | 山东荆卫生物科技有限公司 | Method for detecting absolute content of nicotinamide and nicotinamide mononucleotide |
| WO2023070878A1 (en) * | 2021-10-29 | 2023-05-04 | 深圳先进技术研究院 | Bioluminescence nampt enzyme activity detection composition, kit and detection method |
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| CN103898076A (en) * | 2014-02-24 | 2014-07-02 | 中国科学院武汉物理与数学研究所 | Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor |
| CN103898076B (en) * | 2014-02-24 | 2015-12-02 | 中国科学院武汉物理与数学研究所 | The method of the crosslinked NAMPT of preparation and screening NAMPT inhibitor |
| WO2018023206A1 (en) * | 2016-07-30 | 2018-02-08 | 邦泰生物工程(深圳)有限公司 | Nicotinamide phosphoribosyltransferase mutant and application thereof |
| CN107889505A (en) * | 2016-07-30 | 2018-04-06 | 邦泰生物工程(深圳)有限公司 | A kind of method for preparing nicotinamide mononucleotide |
| CN108026517A (en) * | 2016-07-30 | 2018-05-11 | 邦泰生物工程(深圳)有限公司 | A kind of Nampt mutant and its application |
| US10174298B2 (en) | 2016-07-30 | 2019-01-08 | Bontac Bio-Engineering(Shenzhen) Co., Ltd | Nicotinamide phosphoribosyltransferase (NAMPT) mutant and use thereof |
| CN108026517B (en) * | 2016-07-30 | 2021-05-25 | 邦泰生物工程(深圳)有限公司 | Nicotinamide phosphoribosyl transferase mutant and application thereof |
| CN107889505B (en) * | 2016-07-30 | 2021-05-25 | 邦泰生物工程(深圳)有限公司 | Method for preparing nicotinamide mononucleotide |
| CN114264822A (en) * | 2020-09-16 | 2022-04-01 | 山东荆卫生物科技有限公司 | Method for detecting absolute content of nicotinamide and nicotinamide mononucleotide |
| WO2023070878A1 (en) * | 2021-10-29 | 2023-05-04 | 深圳先进技术研究院 | Bioluminescence nampt enzyme activity detection composition, kit and detection method |
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