CN103898076A - Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor - Google Patents

Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor Download PDF

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CN103898076A
CN103898076A CN201410061169.8A CN201410061169A CN103898076A CN 103898076 A CN103898076 A CN 103898076A CN 201410061169 A CN201410061169 A CN 201410061169A CN 103898076 A CN103898076 A CN 103898076A
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董旭
唐淳
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Wuhan Institute of Physics and Mathematics of CAS
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Abstract

The invention discloses a method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening a NAMPT inhibitor and relates to the field of inhibitors. The method comprises the following steps: preparing a NAMPT recombinant protein; reducing the NAMPT recombinant protein with a reducing agent; carrying out a reaction between the reduced protein and a crosslinking agent so as to obtain the crosslinked NAMPT the active center of which is plugged; compounding a first protein solution and a second protein solution; preparing a first solution to be analyzed, a first contrast solution, a second solution to be analyzed and a second contrast solution; respectively measuring the fluorescence intensities of the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution by using a fluorescence spectrophotometer; and calculating the descending percentage of the fluorescence intensity of the first solution to be analyzed and the second solution to be analyzed. The method comprises few steps, is simple to operate and low in cost; and the external environment hardly influences a reaction process. Thus, the method is good in repeatability and the result is relatively accurate.

Description

The method of the crosslinked NAMPT of preparation and screening NAMPT inhibitor
Technical field
The present invention relates to NAMPT inhibitor and select field, be specifically related to a kind of method of preparing crosslinked NAMPT and screening NAMPT inhibitor.
Background technology
NAMPT(nicotinamidephosphoribosyltransferase, nicotinamide phosphoribosyl transferase) be called again PBEF(pre-B cell colony-enhancing factor, PBEF) or Visfatin(cisceral fat adipokine, Nampt), be NAD(nicotinamde adenine dinucleotide in mammalian body, nicotinamide adenine dinucleotide) remedy the rate-limiting enzyme in synthetic path.NAD is used for participating in the engineerings such as cellular material, energy metabolism, protein modification, DNA reparation, and when human body is in stress situation or produce when disease, the NAD content in human body can increase, and the content of the NAMPT corresponding with NAD also can increase.
NAMPT is extensively distributed in human body, NAMPT (cytoplasm, nucleus and plastosome) in human body cell is different with extracellular mechanism of action, intracellular NAMPT, mainly by synthetic NAD, regulates cellular metabolism, promotes the stable of cytodifferentiation maturation, Promote cell's growth and maintenance cell various functions.Extracellular NAMPT not only can be by synthetic NMN(nicotinamide mononucleotide, nmn) play a role, and itself there is the effect of non-enzyme, be that NAMPT can be served as a cytokine, play a role by acting on its unknown acceptor, as produced the reactions such as transmitting inflammation, oxidative stress.
Research shows, the tumour cell of fast breeding needs more NAD, in tumour cell the content of NAMPT far above normal cell, by the expression of NAMPT in inhibition tumor cell, growth that can inhibition tumor cell.Therefore, NAMPT becomes the novel targets of antitumor drug research.
Existing NAMPT inhibitor comprises that FK866(claims again APO866, English (E)-N-[4-[1-(benzoyl) piperidin-4-yl by name] butyl]-3-pyridin-3-ylprop-2-enamide, molecular formula is C 24h 29n 3o 2) and CB30865 (English name p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+++)-N-(prop-2-ynyl) amino]-N-(3-pyridylmethyl) benzamide, molecular formula C 26h 22brN 5o 2), wherein the antitumor clinical trial of FK866 has been carried out 5, but side effect is larger in use for FK866, and antitumor curative effect is poor, and new NAMPT inhibitor is also in trying to explore.
At present, the method for screening NAMPT inhibitor is following 2 kinds:
(1) carry out medicinal design for the protein conformation of NAMPT: obtain lead compound, it is carried out to structure of modification and obtain inhibitor to be selected; Or obtain parent nucleus by the known inhibitor such as FK866, and parent nucleus is carried out to structure of modification obtain inhibitor to be selected, then by inhibitor to be selected and tumor cell line effect, the good inhibitor to be selected of the screening effect agent finished product that is inhibited.
(2) inhibitor to be selected is added to external NAMPT recombinant protein enzymatic reaction system, obtain NMN(nicotinamide mononucleotide); In NMN, first add methyl phenyl ketone to react, add again acetic acidreaction, obtain the NMN derivative with a fluorophor, NMN derivative is carried out to fluorometric assay, obtain the growing amount of NMN, judge by the growing amount of NMN whether the enzymic activity of NAMPT recombinant protein is subject to the impact of inhibitor to be selected.
But there is respectively following defect in the method for existing screening NAMPT inhibitor:
(1) lead compound and known inhibitor parent nucleus compound structure of modification are obtained to inhibitor to be selected, by the be inhibited method of agent finished product of inhibitor to be selected and tumor cell line effect, can only screen one inhibitor to be selected at every turn, step is more loaded down with trivial details, not only cost compare is high, and working efficiency is lower.
(2) inhibitor to be selected is added to external NAMPT recombinant protein enzymatic reaction system, select the method for inhibitor by measuring NMN, reactions steps is more, operation more complicated, and reaction process is affected by the external environment larger, and detected result accuracy is lower.
Summary of the invention
For the defect existing in prior art, the object of the present invention is to provide a kind of method of preparing crosslinked NAMPT and screening NAMPT inhibitor, cost is lower, working efficiency is higher, and reflection step is less, operates fairly simple, reaction process is affected by the external environment less, and detected result is more accurate.
For reaching above object, the technical scheme that the present invention takes is: a kind of method of preparing crosslinked NAMPT and screening NAMPT inhibitor, it is characterized in that, and comprise the following steps:
S1, preparation NAMPT recombinant protein;
S2, according to molar part, 1 part of NAMPT recombinant protein is added in 20～100 parts of reductive agents; Be to hatch 0.5h～1.5h under the condition of 20 ℃～25 ℃ in temperature, obtain crude protein; Adopt desalting column to remove the reductive agent in crude protein, obtain pre-treatment albumen;
S3, according to molar part, 1 part of pre-treatment albumen is mixed with 2～5 parts of Isosorbide-5-Nitraes-bis-(dimaleoyl imino) butane, be standing and reacting 1h～5h under the condition of 20 ℃～25 ℃ in temperature, adopt desalting column to remove unreacted Isosorbide-5-Nitrae-bis-(dimaleoyl imino) butane, obtain crosslinked NAMPT;
S4, preset PBS buffered soln, be divided into 3 parts by PBS buffered soln: a PBS buffered soln, the 2nd PBS buffered soln, the 3rd PBS buffered soln, and a described PBS buffered soln is identical with the volume of the 2nd PBS buffered soln;
In a PBS buffered soln, add NAMPT recombinant protein, form the first protein solution that concentration is less than 50 μ M; The first protein solution is on average packed in the first silica tube that even number Zhi Rongji is 500 μ L～2mL, described the first silica tube is equally divided into 2 groups: A group, B group;
In the 2nd PBS buffered soln, add crosslinked NAMPT, the second protein solution that configuration concentration is identical with the first protein solution; The second protein solution is on average packed in the second silica tube that even number Zhi Rongji is 500 μ L～2mL, the volume of the second protein solution in every second silica tube, equate with the volume of the first protein solution in every first silica tube, described the second silica tube is equally divided into two groups: C group, D group;
The inhibitor solution to be selected that S5, configuration concentration are 0.5mM～1.5mM, the inhibitor solution to be selected that adds volume to differ in every first silica tube organizing to A, the molar weight of the NAMPT recombinant protein of the first protein solution in every first silica tube: the molar weight of inhibitor solution to be selected is 1:0～1:100; Every first silica tube adds after selecting inhibitor solution and mixes, and is, under the condition of 20 ℃～25 ℃, to leave standstill 5min～15min in temperature, obtains the first solution to be analyzed;
The 3rd PBS buffered soln that adds volume to differ in every first silica tube organizing to B, the volume of the 3rd PBS buffered soln in every first silica tube, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, after adding the 3rd PBS buffered soln, every first silica tube mixes, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min, obtain the first contrast solution;
The solution to be selected that adds volume to differ in every second silica tube organizing to C, the volume of inhibitor solution to be selected in every second silica tube, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, mix, after every second silica tube adds and mixes after selecting inhibitor solution, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min, obtain the second solution to be analyzed;
The 3rd PBS buffered soln that adds volume to differ in every second silica tube organizing to D, the volume of the 3rd PBS buffered soln in every second silica tube, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, every second silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min, obtain the second contrast solution;
S6, use fluorescence spectrophotometer are measured the fluorescence intensity of the first solution to be analyzed, the fluorescence intensity of the first contrast solution, the fluorescence intensity of the second solution to be analyzed, the fluorescence intensity of the second contrast solution;
S7, according to the fluorescence intensity of the fluorescence intensity of the first solution to be analyzed and the first contrast solution, calculate the percentage that the first solution fluorescence intensity to be analyzed declines; According to the fluorescence intensity of the fluorescence intensity of the second solution to be analyzed and the second contrast solution, calculate the percentage that the second solution fluorescence intensity to be analyzed declines.
On the basis of technique scheme, described in inhibitor to be selected be rosmarinic acid, cynarin, 1,3-Dicaffeoylquinic acid or FK866.
On the basis of technique scheme, reductive agent described in step S2 is dithiothreitol (DTT) or three (2-propyloic) phosphine.
On the basis of technique scheme, step S2 comprises the following steps: according to molar part, 1 part of NAMPT recombinant protein being added to concentration is in 30 parts of dithiothreitol (DTT) of 1mM～5mM, be to hatch 1h under the condition of 20 ℃～25 ℃ in temperature, obtain crude protein, adopt desalting column to remove unreacted dithiothreitol (DTT) in crude protein and obtain pre-treatment albumen.
On the basis of technique scheme, step S3 comprises the following steps: by 1, two (dimaleoyl imino) butane of 4-are dissolved in dimethyl sulfoxide (DMSO), to form concentration be 10mM 1, two (dimaleoyl imino) butane solution of 4-, get 120 μ L1, after two (dimaleoyl imino) butane solution of 4-mixes with pre-treatment albumen, be to react 2h under the condition of 20 ℃～25 ℃ in temperature, adopt desalting column to remove unreacted 1, two (dimaleoyl imino) butane of 4-, obtain crosslinked NAMPT.
On the basis of technique scheme, step S4 comprises the following steps: preset PBS buffered soln, PBS buffered soln is divided into 3 parts: a PBS buffered soln, the 2nd PBS buffered soln, the 3rd PBS buffered soln, a described PBS buffered soln is identical with the volume of the 2nd PBS buffered soln;
In a PBS buffered soln, add NAMPT recombinant protein, configuration concentration is the first protein solution of 0.5 μ M; The first protein solution average mark is loaded on to even number and props up in the first silica tube, the first silica tube is equally divided into two groups: A group, B group;
In the 2nd PBS buffered soln, add crosslinked NAMPT, configuration concentration is the second protein solution of 0.5 μ M; The second protein solution splicing is sub-packed in to even number and props up in the second silica tube, the second silica tube is equally divided into two groups: C group, D group.
On the basis of technique scheme, step S5 comprises the following steps: the inhibitor solution to be selected that configuration concentration is 0.5mM, the inhibitor solution to be selected that adds successively volume to differ in the first silica tube of organizing to A, the molar weight of the NAMPT recombinant protein of the first protein solution in every first silica tube: the molar weight of inhibitor solution to be selected is 1:0～1:100; Every first silica tube being added after selecting inhibitor, mix, is under the condition of 20 ℃～25 ℃ in temperature, leaves standstill 10min, obtains the first solution to be analyzed;
The 3rd PBS buffered soln that adds successively volume to differ in the first silica tube of organizing to B, the volume of the 3rd PBS buffered soln and the volume correspondent equal of inhibitor solution to be selected that adds A group, every first silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 10min, obtain the first contrast solution.
On the basis of technique scheme, step S6 comprises the following steps: use fluorescence spectrophotometer, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, the fluorescence intensity that the emission wavelength that detects and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution is 315nm～360nm;
Or use fluorescence spectrophotometer, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, the fluorescence intensity that the emission wavelength that detects and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution is 333nm.
On the basis of technique scheme, the percentile formula that calculates the first solution fluorescence intensity decline to be analyzed described in described step S7 is: first solution fluorescence intensity decline percentage to be analyzed=(fluorescence intensity of fluorescence intensity/the first contrast solution of 1-the first solution to be analyzed) × 100%; The percentile formula that described calculating second solution fluorescence intensity to be analyzed declines is: second solution fluorescence intensity decline percentage to be analyzed=(fluorescence intensity of fluorescence intensity/the second contrast solution of 1-the second solution to be analyzed) × 100%.
On the basis of technique scheme, a kind of for screening the crosslinked NAMPT of NAMPT inhibitor, it is characterized in that: adopt step S1, S2 and S3 in claim 1 to make.
Compared with prior art, the invention has the advantages that:
(1) inhibitor of the present invention reacts with NAMPT recombinant protein and crosslinked NAMPT respectively, obtain the first solution to be analyzed and the second solution to be analyzed, measure the first solution to be analyzed, the first contrast solution, the second solution to be analyzed, the second contrast solution by fluorescence spectrophotometer.
Under the condition that is 280nm at exciting light, the fluorescence intensity that the emission wavelength of generation is 315nm～360nm, or the emission wavelength fluorescence intensity that is 333nm, and the fluorescence intensity of the fluorescence intensity of the first solution to be analyzed and the first contrast solution is compared; The fluorescence intensity of the fluorescence intensity of the second solution to be analyzed and the second contrast solution is compared, judge according to comparison result whether inhibitor has restraining effect to NAMPT recombinant protein and crosslinked NAMPT.
With existing, lead compound and known inhibitor parent nucleus compound structure of modification are obtained to inhibitor to be selected, inhibitor to be selected and tumor cell line effect are inhibited compared with the method for agent finished product, cost compare is low, reflection step is fewer, operate fairly simple, working efficiency is higher, and reaction process is affected by the external environment smaller.
(2) the present invention adopts BMB to react with NAMPT recombinant protein, form activity center by the crosslinked NAMPT of shutoff, the present invention will treat that inhibitor reacts with NAMPT recombinant protein and crosslinked NAMPT respectively, can judge by reaction result whether the action site of inhibitor and NAMPT recombinant protein is positioned at the activity center of NAMPT, if inhibitor to be selected not only can with the effect of NAMPT recombinant protein, and can with crosslinked NAMPT effect, this inhibitor to be selected is NAMPT recombinant protein katalysis inhibitor; If inhibitor to be selected can only with the effect of NAMPT recombinant protein, this inhibitor to be selected may become the non-enzyme function inhibitor of NAMPT.
It is katalysis inhibitor that the present invention can distinguish inhibitor to be selected, or potential non-enzyme function inhibitor, for design and the screening of NAMPT inhibitor provide good instrument.
Accompanying drawing explanation
Fig. 1 is the structural representation of crosslinked NAMPT;
Fig. 2 is that the rosmarinic acid of different molar weights is on the decline trend map of percentile impact of the fluorescence intensity of NAMPT, crosslinked NAMPT;
Fig. 3 is that the cynarin of different molar weights is on the decline trend map of percentile impact of the fluorescence intensity of NAMPT, crosslinked NAMPT;
Fig. 4 is that the 1,3-Dicaffeoylquinic acid of different molar weights is on the decline trend map of percentile impact of the fluorescence intensity of NAMPT, crosslinked NAMPT;
Fig. 5 is that the FK866 of different molar weights is on the decline trend map of percentile impact of the fluorescence intensity of NAMPT, crosslinked NAMPT.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
The method of the crosslinked NAMPT of preparation of the present invention and screening NAMPT inhibitor, comprises the following steps:
S1: preparation NAMPT recombinant protein.
S2: according to molar part, 1 part of NAMPT recombinant protein is added in 20～100 parts of (being preferably 30 parts) reductive agents, reductive agent can be selected DTT(dithiothreitol (DTT)) or TCEP (three (2-propyloic) phosphine); Be under the condition of 20 ℃～25 ℃, to hatch 0.5h～1.5h(to be preferably 1h in temperature), obtain crude protein, adopt desalting column to remove the reductive agent in crude protein, obtain pre-treatment albumen;
S3: according to molar part by 1 part of pre-treatment albumen and 2～5 parts of cross-linking reagent BMB(1, two (dimaleoyl imino) butane of 4-) add reactor, mix, be under the condition of 20 ℃～25 ℃, to react 1h～5h(to be preferably 2h in temperature) after, adopt desalting column to remove unreacted BMB, obtain crosslinked NAMPT;
S4: preset PBS buffered soln, is divided into three parts by PBS buffered soln: a PBS buffered soln, the 2nd PBS buffered soln and the 3rd PBS buffered soln, wherein, the volume of a PBS buffered soln and the 2nd PBS buffered soln equates.
In a PBS buffered soln, add NAMPT recombinant protein, form concentration and be less than 50 μ M(and be preferably 0.5 μ M) the first protein solution; The first protein solution is on average packed in the first silica tube that even number Zhi Rongji is 500 μ l～2mL, even number is propped up to the first silica tube and be equally divided into 2 groups: A group, B group;
In the 2nd PBS buffered soln, add crosslinked NAMPT, the second protein solution that configuration concentration is identical with the first protein solution; The second protein solution is on average packed in the second silica tube that even number Zhi Rongji is 500 μ l～2mL, the volume of the second albumen in every second silica tube, equate with the volume of the first albumen in every first silica tube, even number is propped up to the second silica tube and be equally divided into two groups: C group, D group;
The inhibitor solution to be selected that S5, configuration concentration are 0.5mM～1.5mM, inhibitor to be selected can be selected rosmarinic acid, cynarin, 1,3-Dicaffeoylquinic acid or FK866.The inhibitor solution to be selected that adds successively volume to differ in the first silica tube of organizing to A, the molar weight of the NAMPT recombinant protein of the first protein solution in every first silica tube: the molar weight of inhibitor solution to be selected is 1:0～1:100; In every first silica tube, adding after selecting inhibitor solution and mix, is under the condition of 20 ℃～25 ℃ in temperature, leaves standstill 5min～15min(and is preferably 10min), obtain the first solution to be analyzed;
The 3rd PBS buffered soln that adds successively volume to differ in all the first silica tubes of organizing to B, the volume of the 3rd PBS buffered soln in every first silica tube of B group, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, after adding the 3rd PBS buffered soln in every first silica tube, mix, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min(and be preferably 10min), obtain the first contrast solution;
The inhibitor solution to be selected that adds successively volume to differ in all the second silica tubes of organizing to C, the volume of inhibitor solution to be selected in every second silica tube of C group, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, in every second silica tube, add after selecting inhibitor solution and mix, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min(and be preferably 10min), obtain the second solution to be analyzed;
The 3rd PBS buffered soln that adds successively volume to differ in all the second silica tubes of organizing to D, the volume of the 3rd PBS buffered soln in every second silica tube of D group, identical with the volume of inhibitor solution to be selected in the second silica tube corresponding to C group, after adding the 3rd PBS buffered soln, every second silica tube mixes, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min(and be preferably 10min), obtain the second contrast solution;
S6: use the fluorescence spectrophotometer that model is HORIBA FM4, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, detect and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution fluorescence intensity that emission wavelength is 315nm～360nm respectively.
Or use the fluorescence spectrophotometer that model is HORIBA FM4, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, detect and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution fluorescence intensity that emission wavelength is 333nm respectively.
S7, according to the fluorescence intensity of the fluorescence intensity of the first solution to be analyzed and the first contrast solution, calculate the percentage that the first solution fluorescence intensity to be analyzed declines; According to the fluorescence intensity of the fluorescence intensity of the second solution to be analyzed and the second contrast solution, calculate the percentage that the second solution fluorescence intensity to be analyzed declines.
In the embodiment of the present invention, preparing NAMPT recombinant protein comprises the following steps:
By pET-11a-hnampt (G355C, D393C) be all converted into e. coli bl21 (DE3) Star competence bacterium, competence bacterium is applied to the LB flat board that contains 50 μ g/ml penbritins (aseptic, be set in culture dish, mixed the solid medium of agar) upper surface, under the aseptic condition of 37 ℃, cultivate after 12h, by colony lift to the LB substratum that contains 50 μ g/ml penbritins, the OD600=0.6(OD that is cultured to clone bacterium liquid under the condition of 37 ℃ refers to the light absorption value of certain solution at 600nm wavelength place), adding concentration is the IPTG(isopropylthiogalactoside of 0.5mmol/L), under the condition of 20 ℃, induced reaction 12h obtains bacterial culture fluid.
Get 10000g bacterial culture fluid and in temperature be centrifugal 10min under the condition of 4 ℃, collect bacterial precipitation, with the resuspended bacterium of lysate, adopting model is the pressure breaking instrument of avestin EmulsiFlex-C3, under the condition that is 15000bar at pressure, smudge cells obtains cell suspending liquid, be centrifugal 30min under 4 ℃, the rotating speed condition that is 20000rpm by cell suspending liquid in temperature, collect supernatant liquor.
Adopt protein purification chromatographic system to carry out protein purification to supernatant liquor, obtain purifying protein; Select HisTrap FF crude chromatography column, the imidazoles solution that working concentration is 30mM～300mM carries out gradient elution to purifying protein, and collecting ultraviolet absorption peak is the first elutriant that 280nm is corresponding.
Adopt the molecular sieve that model is S-200 to carry out purifying to the first elutriant, collect the effluent liquid that exclusion volume is 130ml left and right; Adopting model is the anion-exchange column of Source Q, and selecting concentration is that the NaCl solution of 0mM～500mM carries out gradient elution to effluent liquid, and collecting ultraviolet absorption peak is the second elutriant that 280nm is corresponding, obtains NAMPT recombinant protein.
Reaction mechanism of the present invention is as follows:
In the embodiment of the present invention, NAMPT recombinant protein is two NAMPT(G355C and D393C) monomer form NAMPT dimer, this dimer has two catalytic centers, all there are two pairs of halfcystines the ingress of each catalytic center, NAMPT recombinant protein reacts with DTT, two pairs of halfcystines of NAMPT recombinant protein are all reduced into cysteinyl, form pre-treatment albumen.Pre-treatment albumen reacts with BMB; the acyl group effect of cysteinyl in BMB and pre-treatment albumen; form crosslinked NAMPT; shown in Figure 1; green ribbon is BMB; red site is BMB and NAMPT crosslink sites, and crosslinked part is the catalytic center being reduced that is positioned at NAMPT recombinant protein, and crosslinked NAMPT is that catalytic center is by the NAMPT recombinant protein of BMB shutoff.
The embodiment of the present invention by measure NAMPT recombinant protein respectively with fluorescence intensity after selecting inhibitor, the effect of PBS buffered soln, thereby determine the impact of inhibitor to be selected on NAMPT recombinant protein; The embodiment of the present invention by will inhibitor be selected respectively at NAMPT recombinant protein, crosslinked NAMPT effect, thereby the binding site of definite inhibitor to be selected and NAMPT recombinant protein, and the bonding strength of inhibitor to be selected and NAMPT recombinant protein.
The embodiment of the present invention is that the variation by measuring NAMPT recombinant protein endogenous fluorescence realizes, and cost compare is low, not only operates fairly simplely, and the single measurement time is shorter.
In connection with 4 embodiment, the present invention is made to detailed description below.
Embodiment 1, selecting inhibitor to be selected is rosmarinic acid.
S1: preparation NAMPT recombinant protein.
S2: it is in the DTT (dithiothreitol (DTT)) of 5mM that the NAMPT recombinant protein that is 200 μ M by 2mL concentration adds 1.6mL concentration, be, under the condition of 20 ℃, to hatch 1h in temperature, obtain crude protein, adopt desalting column to remove the DTT in crude protein, obtain pre-treatment albumen.
S3: the BMB cross-linking reagent that is 10mM by pre-treatment albumen and 80 μ L concentration adds test tube, mixes, is to react after 2h under the condition of 20 ℃ in temperature, removes unnecessary BMB with desalting column, obtains crosslinked NAMPT.
S4: preset PBS buffered soln, PBS buffered soln is divided into three parts: a PBS buffered soln, the 2nd PBS buffered soln and the 3rd PBS buffered soln, wherein, the one PBS buffered soln is identical with the volume of the 2nd PBS buffered soln, in a PBS buffered soln, add NAMPT recombinant protein, forming concentration is the first protein solution of 0.5 μ M, and the first protein solution is on average packed in the first silica tube that 30 volumes are 2mL, the first silica tube is equally divided into two groups: A group, B group.
In the 2nd PBS buffered soln, add crosslinked NAMPT, configuration concentration is the second protein solution of 0.5 μ M, and the first protein solution is on average packed in the second silica tube that 30 volumes are 2mL, the second silica tube is equally divided into two groups: C group, D group.
S5: configuration concentration is 1mM rosmarinic acid adds rosmarinic acid: in first the first silica tube, add 0 μ L rosmarinic acid in the first silica tube of A group, in second the first silica tube, add 0.5 μ L rosmarinic acid, in the 3rd the first silica tube, add 1 μ L rosmarinic acid, in the 4th the first silica tube, add 1.5 μ L rosmarinic acids, in the 5th the first silica tube, add 2 μ L rosmarinic acids, in the 6th the first silica tube, add 3 μ L rosmarinic acids, in the 7th the first silica tube, add 4 μ L rosmarinic acids, in the 8th the first silica tube, add 5 μ L rosmarinic acids, in the 9th the first silica tube, add 7 μ L rosmarinic acids, in the tenth the first silica tube, add 10 μ L rosmarinic acids, in the 11 the first silica tube, add 15 μ L rosmarinic acids, in twelve earthly branches the first silica tube, add 20 μ L rosmarinic acids, in the 13 the first silica tube, add 25 μ L rosmarinic acids, in the 14 the first silica tube, add 35 μ L rosmarinic acids, in the 15 the first silica tube, add 50 μ L rosmarinic acids.Every first silica tube adds after rosmarinic acid, mixes, and is under the condition of 20 ℃, to leave standstill 10min in temperature, obtains the first analytical solution.
In the first silica tube of B group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L, after adding the 3rd PBS buffered soln, every first silica tube mixes, be under the condition of 20 ℃, to leave standstill 10min in temperature, obtain the first contrast solution.
In every second silica tube organizing to C, adding concentration is 1mM rosmarinic acid: the volume of dropping is followed successively by 0 μ L, 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L.Every second silica tube adds after rosmarinic acid, mixes, and is under the condition of 20 ℃, to leave standstill 10min in temperature, obtains the second analytical solution.
In the second silica tube of D group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L, every second silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 10min in temperature, obtain the second contrast solution.
S6: use the fluorescence spectrophotometer that model is HORIBA FM4, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, the fluorescence intensity that the emission wavelength of measuring and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution is 315nm～360nm;
S7: percentage=(fluorescence intensity of fluorescence intensity/the first contrast solution of 1-the first solution to be analyzed) × 100% that calculates the fluorescence intensity decline of the first solution to be measured; Calculate percentage=(fluorescence intensity of fluorescence intensity/the second contrast solution of 1-the second solution to be analyzed) × 100% of the fluorescence intensity decline of the second solution to be measured.
Shown in Figure 2, be fluorescence intensity percentage and the graph of a relation that adds rosmarinic acid molar weight of NAMPT recombinant protein, and the fluorescence intensity percentage of crosslinked NAMPT and the trend map that adds rosmarinic acid molar weight.
Rosmarinic acid adds after NAMPT recombinant protein, the fluorescence intensity of NAMPT recombinant protein declines to some extent, along with the increase of rosmarinic acid add-on, the fluorescence decline percentage of NAMPT recombinant protein reduces more obvious, illustrate rosmarinic acid can with the effect of NAMPT recombinant protein.
Rosmarinic acid adds after crosslinked NAMPT, the fluorescence intensity of crosslinked NAMPT declines to some extent, rosmarinic acid add-on is more, the fluorescence decline percentage of crosslinked NAMPT reduces more obvious, illustrate rosmarinic acid can with crosslinked NAMPT effect, because crosslinked NAMPT is that catalytic center is by the NAMPT recombinant protein of shutoff, therefore can illustrate: rosmarinic acid can not interact with NAMPT recombinant protein catalytic center, but rosmarinic acid can be incorporated into the site combination beyond NAMPT recombinant protein catalytic center, rosmarinic acid can be as the non-enzyme function inhibitor of NAMPT.
Embodiment 2: selecting inhibitor to be selected is cynarin.
S1: preparation NAMPT recombinant protein.
S2: it is in the ECET of 1mM that the NAMPT recombinant protein that is 200 μ M by 3mL concentration adds 36mL concentration, is, under the condition of 25 ℃, to hatch 0.5h in temperature, obtains crude protein, and the DTT that adopts desalting column to remove in crude protein obtains pre-treatment albumen.
S3: the BMB that is 10mM by pre-treatment albumen and 240 μ L concentration adds test tube, mixes, is to react after 1h under the condition of 20 ℃ in temperature, removes unnecessary BMB with desalting column, obtains crosslinked NAMPT.
S4: preset PBS buffered soln, PBS buffered soln is divided into three parts: a PBS buffered soln, the 2nd PBS buffered soln and the 3rd PBS buffered soln, wherein, the one PBS buffered soln is identical with the volume of the 2nd PBS buffered soln, in a PBS buffered soln, add NAMPT recombinant protein, forming concentration is the first protein solution of 1 μ M, and the first protein solution is on average enclosed in the first silica tube that 20 volumes are 5mL, 20 the first silica tubes is equally divided into two groups: A group, B group.
In the 2nd PBS buffered soln, add crosslinked NAMPT, configuration concentration is the second protein solution of 1 μ M, and the second protein solution is divided in the second silica tube that 20 volumes are 5mL, 20 the second silica tubes is equally divided into two groups: C group, D group.
S5: configuration concentration is 2mM cynarin, in the first silica tube of A group, add cynarin successively: in first the first silica tube, add 0 μ L cynarin, in second the first silica tube, add 1.5 μ L cynarins, in the 3rd the first silica tube, add 3 μ L cynarins, in the 4th the first silica tube, add 5 μ L cynarins, in the 5th the first silica tube, add 7 μ L cynarins, in the 6th the first silica tube, add 10 μ L cynarins, in the 7th the first silica tube, add 20 μ L cynarins, in the 8th the first silica tube, add 25 μ L cynarins, in the 9th the first silica tube, add 35 μ L cynarins, in the tenth the first silica tube, add 50 μ L cynarins.Every first silica tube adds after cynarin, mixes, and be the condition standing and reacting 15min of 20 ℃ in temperature, obtain the first analytical solution.
In the first silica tube of B group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 1.5 μ L, 3 μ L, 5 μ L, 7 μ L, 10 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L, every first silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 15min in temperature, obtain the first contrast solution.
In the second silica tube of C group, add cynarin: the volume of dropping is followed successively by 0 μ L, 1.5 μ L, 3 μ L, 5 μ L, 7 μ L, 10 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L.Every second silica tube adds after cynarin, mixes, and is under the condition of 20 ℃, to leave standstill 15min in temperature, obtains the second analytical solution.
In the second silica tube of D group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 1.5 μ L, 3 μ L, 5 μ L, 7 μ L, 10 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L, every second silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 15min in temperature, obtain the second contrast solution.
S6: use the fluorescence spectrophotometer that model is HORIBA FM4, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, measure and record the fluorescence intensity that the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution are 333nm at emission wavelength.
S7: percentage=(fluorescence intensity of fluorescence intensity/the first contrast solution of 1-the first solution to be analyzed) × 100% that calculates the fluorescence intensity decline of the first solution to be measured; Calculate percentage=(fluorescence intensity of fluorescence intensity/the second contrast solution of 1-the second solution to be analyzed) × 100% of the fluorescence intensity decline of the second solution to be measured.
Shown in Figure 3, be fluorescence intensity percentage and the graph of a relation that adds cynarin molar weight of NAMPT recombinant protein, and the fluorescence intensity percentage of crosslinked NAMPT recombinant protein and the trend map that adds cynarin molar weight.
Cynarin adds after NAMPT recombinant protein, and the fluorescence intensity of NAMPT recombinant protein declines to some extent, and along with the increase of cynarin add-on, the fluorescence decline percentage of NAMPT recombinant protein reduces more obvious, illustrate cynarin can with the effect of NAMPT recombinant protein.
Cynarin adds after crosslinked NAMPT, the fluorescence intensity of crosslinked NAMPT declines to some extent, cynarin add-on is more, the fluorescence decline percentage of crosslinked NAMPT reduces more obvious, illustrate cynarin can with crosslinked NAMPT effect, because crosslinked NAMPT is that catalytic center is by the NAMPT recombinant protein of shutoff, therefore can illustrate: cynarin can not interact with NAMPT catalytic center, but can be in conjunction with the site combination beyond NAMPT catalytic center, cynarin can be as the non-enzyme function inhibitor of NAMPT.
Embodiment 3: selecting inhibitor to be selected is 1,3-Dicaffeoylquinic acid.
S1: preparation NAMPT recombinant protein.
S2: it is in the 1mol dithiothreitol (DTT) of 5mM that the NAMPT recombinant protein that is 200 μ M by 4mL concentration adds concentration, hatches 1.5h, obtains crude protein, the DTT that adopts desalting column to remove in crude protein obtains pre-treatment albumen.
S3: the BMB that is 10mM by pre-treatment albumen and 160 μ L concentration adds test tube, mixes, is, under the condition of 22 ℃, after reaction 5h, to remove unnecessary BMB with desalting column in temperature, obtains crosslinked NAMPT.
S4: preset PBS buffered soln, PBS buffered soln is divided into three parts: a PBS buffered soln, the 2nd PBS buffered soln and the 3rd PBS buffered soln, wherein, the one PBS buffered soln is identical with the volume of the 2nd PBS buffered soln, in a PBS buffered soln, add NAMPT, configuration concentration is the first protein solution of 0.5 μ M, and the first protein solution is on average packed in the first silica tube that 24 volumes are 1mL, the first silica tube is equally divided into two groups: A group, B group.
In the 2nd PBS buffered soln, add crosslinked NAMPT, configuration concentration is the second protein solution of 0.5 μ M, and the second protein solution is on average packed in the second silica tube that 24 volumes are 1mL, the second silica tube is equally divided into two groups: C group, D group.
S5: configuration concentration is 2mM1, 3-dicaffeoylquinic acid solution, in every first silica tube of A group, add 1, 3-dicaffeoylquinic acid solution: add 0 μ L1 in first the first silica tube, 3-dicaffeoylquinic acid solution, in second the first silica tube, add 0.25 μ L1, 3-dicaffeoylquinic acid solution, in the 3rd the first silica tube, add 0.5 μ L1, 3-dicaffeoylquinic acid solution, in the 4th the first silica tube, add 1 μ L1, 3-dicaffeoylquinic acid solution, in the 5th the first silica tube, add 1.25 μ L1, 3-dicaffeoylquinic acid solution, in the 6th the first silica tube, add 1.75 μ L1, 3-dicaffeoylquinic acid solution, in the 7th the first silica tube, add 2.5 μ L1, 3-dicaffeoylquinic acid solution, in the 8th the first silica tube, add 3.75 μ L1, 3-dicaffeoylquinic acid solution, in the 9th the first silica tube, add 5 μ L1, 3-dicaffeoylquinic acid solution, in the tenth the first silica tube, add 5.25 μ L1, 3-dicaffeoylquinic acid solution, in the 11 the first silica tube, add 8.75 μ L1, 3-dicaffeoylquinic acid solution, in twelve earthly branches the first silica tube, add 12.5 μ L1, 3-dicaffeoylquinic acid solution.After every first silica tube adds 1,3-Dicaffeoylquinic acid solution, all shaking up, is under the condition of 25 ℃, to leave standstill 10min in temperature, obtains the first analytical solution.
In every first silica tube of B group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 0.25 μ L, 0.5 μ L, 1 μ L, 1.25 μ L, 1.75 μ L, 2.5 μ L, 3.75 μ L, 5 μ L, 5.25 μ L, 8.75 μ L and 12.5 μ L, in every first silica tube, add after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 10min in temperature, obtain the first contrast solution.
In every second silica tube of C group, add 1,3-Dicaffeoylquinic acid solution: the volume of dropping is followed successively by 0 μ L, 0.25 μ L, 0.5 μ L, 1 μ L, 1.25 μ L, 1.75 μ L, 2.5 μ L, 3.75 μ L, 5 μ L, 5.25 μ L, 8.75 μ L and 12.5 μ L.In every second silica tube, adding after 1,3-Dicaffeoylquinic acid solution, mix, is under the condition of 20 ℃, to leave standstill 10min in temperature, obtains the second analytical solution.
In the second silica tube of D group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 0.25 μ L, 0.5 μ L, 1 μ L, 1.25 μ L, 1.75 μ L, 2.5 μ L, 3.75 μ L, 5 μ L, 5.25 μ L, 8.75 μ L and 12.5 μ L, every second silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 10min in temperature, obtain the second contrast solution.
S6: use the fluorescence spectrophotometer that model is HORIBA FM4, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, detect and record the fluorescence intensity that the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution are 315nm～360nm at emission wavelength.
S7: percentage=(fluorescence intensity of fluorescence intensity/the first contrast solution of 1-the first solution to be analyzed) × 100% that calculates the fluorescence intensity decline of the first solution to be measured; Calculate percentage=(fluorescence intensity of fluorescence intensity/the second contrast solution of 1-the second solution to be analyzed) × 100% of the fluorescence intensity decline of the second solution to be measured.
Shown in Figure 4, be fluorescence intensity percentage and the graph of a relation that adds 1,3-Dicaffeoylquinic acid molar weight of NAMPT recombinant protein, and the fluorescence intensity percentage of crosslinked NAMPT and the trend map that adds 1,3-Dicaffeoylquinic acid molar weight.
1,3-dicaffeoylquinic acid adds after NAMPT recombinant protein, the fluorescence intensity of NAMPT recombinant protein declines to some extent, along with 1, the increase of 3-dicaffeoylquinic acid add-on, the fluorescence decline percentage of NAMPT recombinant protein reduces more obvious, illustrate 1,3-Dicaffeoylquinic acid can with NAMPT effect.
1, 3-dicaffeoylquinic acid adds after crosslinked NAMPT, the fluorescence intensity of crosslinked NAMPT declines to some extent, 1, 3-dicaffeoylquinic acid add-on is more, the fluorescence decline percentage of crosslinked NAMPT reduces more obvious, illustrate 1, 3-dicaffeoylquinic acid can with crosslinked NAMPT effect, because crosslinked NAMPT is that catalytic center is by the NAMPT recombinant protein of shutoff, therefore can illustrate: 1, 3-dicaffeoylquinic acid can not interact with NAMPT recombinant protein catalytic center, but can be combined with the site beyond NAMPT catalytic center, 1, 3-dicaffeoylquinic acid can be as the non-enzyme function inhibitor of NAMPT.
Embodiment 4, selecting inhibitor to be selected is FK866.
S1: preparation NAMPT recombinant protein.
S2: it is in the DTT (dithiothreitol (DTT)) of 5mM that the NAMPT recombinant protein that is 200 μ M by 2mL concentration adds 1.6mL concentration, hatches 1h, obtains crude protein, the DTT that adopts desalting column to remove in crude protein obtains pre-treatment albumen.
S3: the BMB cross-linking reagent that is 10mM by pre-treatment albumen and 80 μ L concentration adds test tube, mixes, is, under the condition of 25 ℃, after reaction 2h, to remove unnecessary BMB with desalting column in temperature, obtains crosslinked NAMPT.
S4: preset PBS buffered soln, PBS buffered soln is divided into three parts: a PBS buffered soln, the 2nd PBS buffered soln and the 3rd PBS buffered soln, wherein, the one PBS buffered soln is identical with the volume of the 2nd PBS buffered soln, in a PBS buffered soln, add NAMPT recombinant protein, configuration concentration is the first protein solution of 0.5 μ M, and the first protein solution is on average packed in the first silica tube that 30 volumes are 2mL, the first silica tube is equally divided into two groups: A group, B group.
In the 2nd PBS buffered soln, add crosslinked NAMPT, configuration concentration is the second protein solution of 0.5 μ M, and the second protein solution is on average packed in the second silica tube that 30 volumes are 2mL, the second silica tube is equally divided into two groups: C group, D group.
S5: configuration concentration is 1mMFK866 adds FK866: in first the first silica tube liquid, add 0mLFK866 in the first silica tube of A group, in second the first silica tube, add 0.5 μ LFK866, in the 3rd the first silica tube, add 1 μ LFK866, in the 4th the first silica tube, add 1.5 μ LFK866, in the 5th the first silica tube, add 2 μ LFK866, in the 6th the first silica tube, add 3 μ LFK866, in the 7th the first silica tube, add 4 μ LFK866, in the 8th the first silica tube, add 5 μ LFK866, in the 9th the first silica tube, add 7 μ LFK866, in the tenth the first silica tube, add 10 μ LFK866, in the 11 the first silica tube, add 15 μ LFK866, in twelve earthly branches the first silica tube, add 20 μ LFK866, in the 13 the first silica tube, add 25 μ LFK866, in the 14 the first silica tube, add 35 μ LFK866, in the 15 the first silica tube, add 50 μ LFK866.Every first silica tube adds after FK866, mixes, and is under the condition of 20 ℃, to leave standstill 10min in temperature, obtains the first analytical solution.
In every part of first protein solution of B group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L, every first silica tube adds after PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 10min in temperature, obtain the first contrast solution.
In every part of second protein solution of C group, add FK866: the volume of dropping is followed successively by 0 μ L, 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L.Every second silica tube adds after FK866, mixes, and is under the condition of 20 ℃, to leave standstill 10min in temperature, obtains the second analytical solution.
In every part of second protein solution of D group, drip the 3rd PBS buffered soln, the volume dripping is followed successively by 0 μ L, 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 35 μ L and 50 μ L, every second silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃, to leave standstill 10min in temperature, obtain the second contrast solution.
S6: use the fluorescence spectrophotometer that model is HORIBA FM4, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, detect and record the fluorescence intensity that the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution are 315nm～360nm at emission wavelength;
S7: percentage=(fluorescence intensity of fluorescence intensity/the first contrast solution of 1-the first solution to be analyzed) × 100% that calculates the fluorescence intensity decline of the first solution to be measured; Calculate percentage=(fluorescence intensity of fluorescence intensity/the second contrast solution of 1-the second solution to be analyzed) × 100% of the fluorescence intensity decline of the second solution to be measured.
Shown in Figure 5, be fluorescence intensity percentage and the graph of a relation that adds FK866 molar weight of NAMPT recombinant protein, and the fluorescence intensity percentage of crosslinked NAMPT recombinant protein and the trend map that adds FK866 molar weight.
FK866 adds after NAMPT recombinant protein, and the fluorescence intensity of NAMPT recombinant protein declines to some extent, and along with the increase of FK866 add-on, the fluorescence decline percentage of NAMPT recombinant protein reduces more obvious, illustrate FK866 can with the effect of NAMPT recombinant protein.
FK866 adds after crosslinked NAMPT, it is stable that the fluorescence intensity of crosslinked NAMPT keeps, the fluorescence decline percentage of crosslinked NAMPT does not reduce, illustrate FK866 can not with crosslinked NAMPT effect, therefore can illustrate: FK866 can only be combined with the catalytic center of NAMPT recombinant protein, can not be combined with other site of NAMPT recombinant protein.FK866 is a known NAMPT katalysis inhibitor, and the method that adopts us to set up obtains result and conforms to therewith.
The present invention is not limited to above-mentioned embodiment, for those skilled in the art, under the premise without departing from the principles of the invention, can also make some improvements and modifications, within these improvements and modifications are also considered as protection scope of the present invention.The content not being described in detail in this specification sheets belongs to the known prior art of professional and technical personnel in the field.

Claims (10)

1. a method of preparing crosslinked NAMPT and screening NAMPT inhibitor, is characterized in that, comprises the following steps:
S1, preparation NAMPT recombinant protein;
S2, according to molar part, 1 part of NAMPT recombinant protein is added in 20～100 parts of reductive agents; Be to hatch 0.5h～1.5h under the condition of 20 ℃～25 ℃ in temperature, obtain crude protein; Adopt desalting column to remove the reductive agent in crude protein, obtain pre-treatment albumen;
S3, according to molar part, 1 part of pre-treatment albumen is mixed with 2～5 parts of Isosorbide-5-Nitraes-bis-(dimaleoyl imino) butane, be standing and reacting 1h～5h under the condition of 20 ℃～25 ℃ in temperature, adopt desalting column to remove unreacted Isosorbide-5-Nitrae-bis-(dimaleoyl imino) butane, obtain crosslinked NAMPT;
S4, preset PBS buffered soln, be divided into 3 parts by PBS buffered soln: a PBS buffered soln, the 2nd PBS buffered soln, the 3rd PBS buffered soln, and a described PBS buffered soln is identical with the volume of the 2nd PBS buffered soln;
In a PBS buffered soln, add NAMPT recombinant protein, form the first protein solution that concentration is less than 50 μ M; The first protein solution is on average packed in the first silica tube that even number Zhi Rongji is 500 μ L～2mL, described the first silica tube is equally divided into 2 groups: A group, B group;
In the 2nd PBS buffered soln, add crosslinked NAMPT, the second protein solution that configuration concentration is identical with the first protein solution; The second protein solution is on average packed in the second silica tube that even number Zhi Rongji is 500 μ L～2mL, the volume of the second protein solution in every second silica tube, equate with the volume of the first protein solution in every first silica tube, described the second silica tube is equally divided into two groups: C group, D group;
The inhibitor solution to be selected that S5, configuration concentration are 0.5mM～1.5mM, the inhibitor solution to be selected that adds volume to differ in every first silica tube organizing to A, the molar weight of the NAMPT recombinant protein of the first protein solution in every first silica tube: the molar weight of inhibitor solution to be selected is 1:0～1:100; Every first silica tube adds after selecting inhibitor solution and mixes, and is, under the condition of 20 ℃～25 ℃, to leave standstill 5min～15min in temperature, obtains the first solution to be analyzed;
The 3rd PBS buffered soln that adds volume to differ in every first silica tube organizing to B, the volume of the 3rd PBS buffered soln in every first silica tube, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, after adding the 3rd PBS buffered soln, every first silica tube mixes, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min, obtain the first contrast solution;
The solution to be selected that adds volume to differ in every second silica tube organizing to C, the volume of inhibitor solution to be selected in every second silica tube, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, mix, after every second silica tube adds and mixes after selecting inhibitor solution, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min, obtain the second solution to be analyzed;
The 3rd PBS buffered soln that adds volume to differ in every second silica tube organizing to D, the volume of the 3rd PBS buffered soln in every second silica tube, identical with the volume of inhibitor solution to be selected in the first silica tube corresponding to A group, every second silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 5min～15min, obtain the second contrast solution;
S6, use fluorescence spectrophotometer are measured the fluorescence intensity of the first solution to be analyzed, the fluorescence intensity of the first contrast solution, the fluorescence intensity of the second solution to be analyzed, the fluorescence intensity of the second contrast solution;
S7, according to the fluorescence intensity of the fluorescence intensity of the first solution to be analyzed and the first contrast solution, calculate the percentage that the first solution fluorescence intensity to be analyzed declines; According to the fluorescence intensity of the fluorescence intensity of the second solution to be analyzed and the second contrast solution, calculate the percentage that the second solution fluorescence intensity to be analyzed declines.
2. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, is characterized in that: described in inhibitor to be selected be rosmarinic acid, cynarin, 1,3-Dicaffeoylquinic acid or FK866.
3. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, is characterized in that: reductive agent described in step S2 is dithiothreitol (DTT) or three (2-propyloic) phosphine.
4. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, it is characterized in that, step S2 comprises the following steps: according to molar part, 1 part of NAMPT recombinant protein being added to concentration is in 30 parts of dithiothreitol (DTT) of 1mM～5mM, be to hatch 1h under the condition of 20 ℃～25 ℃ in temperature, obtain crude protein, adopt desalting column to remove unreacted dithiothreitol (DTT) in crude protein and obtain pre-treatment albumen.
5. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, it is characterized in that, step S3 comprises the following steps: by 1, two (dimaleoyl imino) butane of 4-are dissolved in dimethyl sulfoxide (DMSO), to form concentration be 10mM 1, two (dimaleoyl imino) butane solution of 4-, get 120 μ L1, after two (dimaleoyl imino) butane solution of 4-mixes with pre-treatment albumen, be to react 2h under the condition of 20 ℃～25 ℃ in temperature, adopt desalting column to remove unreacted 1, two (dimaleoyl imino) butane of 4-, obtain crosslinked NAMPT.
6. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, it is characterized in that, step S4 comprises the following steps: preset PBS buffered soln, PBS buffered soln is divided into 3 parts: a PBS buffered soln, the 2nd PBS buffered soln, the 3rd PBS buffered soln, a described PBS buffered soln is identical with the volume of the 2nd PBS buffered soln;
In a PBS buffered soln, add NAMPT recombinant protein, configuration concentration is the first protein solution of 0.5 μ M; The first protein solution average mark is loaded on to even number and props up in the first silica tube, the first silica tube is equally divided into two groups: A group, B group;
In the 2nd PBS buffered soln, add crosslinked NAMPT, configuration concentration is the second protein solution of 0.5 μ M; The second protein solution splicing is sub-packed in to even number and props up in the second silica tube, the second silica tube is equally divided into two groups: C group, D group.
7. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, it is characterized in that, step S5 comprises the following steps: the inhibitor solution to be selected that configuration concentration is 0.5mM, the inhibitor solution to be selected that adds successively volume to differ in the first silica tube of organizing to A, the molar weight of the NAMPT recombinant protein of the first protein solution in every first silica tube: the molar weight of inhibitor solution to be selected is 1:0～1:100; Every first silica tube being added after selecting inhibitor, mix, is under the condition of 20 ℃～25 ℃ in temperature, leaves standstill 10min, obtains the first solution to be analyzed;
The 3rd PBS buffered soln that adds successively volume to differ in the first silica tube of organizing to B, the volume of the 3rd PBS buffered soln and the volume correspondent equal of inhibitor solution to be selected that adds A group, every first silica tube adds after the 3rd PBS buffered soln, mix, be under the condition of 20 ℃～25 ℃ in temperature, leave standstill 10min, obtain the first contrast solution.
8. the method for the crosslinked NAMPT of preparation as claimed in claim 1 and screening NAMPT inhibitor, it is characterized in that, step S6 comprises the following steps: use fluorescence spectrophotometer, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, the fluorescence intensity that the emission wavelength that detects and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution is 315nm～360nm;
Or use fluorescence spectrophotometer, under the condition that is 280nm in excitation wavelength, excite respectively the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution, the fluorescence intensity that the emission wavelength that detects and record the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution is 333nm.
9. the method for the crosslinked NAMPT of the preparation as described in claim 1～8 any one and screening NAMPT inhibitor, is characterized in that: the percentile formula that calculates the first solution fluorescence intensity decline to be analyzed described in described step S7 is: first solution fluorescence intensity decline percentage to be analyzed=(fluorescence intensity of fluorescence intensity/the first contrast solution of 1-the first solution to be analyzed) × 100%; The percentile formula that described calculating second solution fluorescence intensity to be analyzed declines is: second solution fluorescence intensity decline percentage to be analyzed=(fluorescence intensity of fluorescence intensity/the second contrast solution of 1-the second solution to be analyzed) × 100%.
10. for screening a crosslinked NAMPT for NAMPT inhibitor, it is characterized in that: adopt step S1, S2 and S3 in claim 1 to make.
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