WO2023070878A1

WO2023070878A1 - Bioluminescence nampt enzyme activity detection composition, kit and detection method

Info

Publication number: WO2023070878A1
Application number: PCT/CN2021/138125
Authority: WO
Inventors: 王沛; 於邱黎阳
Original assignee: 深圳先进技术研究院
Priority date: 2021-10-29
Filing date: 2021-12-14
Publication date: 2023-05-04
Also published as: CN116064721A

Abstract

A bioluminescence NAMPT enzyme activity detection composition, a kit and a detection method. The bioluminescence detection composition comprises NAM, PRPP, ATP, NMNAT and NAD+ bioluminescence probes and corresponding bioluminescence probe substrates. The detection method comprises: performing luminescence intensity detection on a sample to be detected by using the bioluminescence detection composition or kit, calculating the NAD+ concentration, and using linear regression to determine the NAD+ generation rate, or NAMPT enzyme activity, in a reaction system. The direct, real-time and interference-free measurement of the NAD+ concentration in a NAMPT enzyme activity detection system is realized. An unpurified physiological sample can be directly detected.

Description

A bioluminescent detection composition, kit and detection method for NAMPT enzyme activity

technical field

The invention belongs to the field of biotechnology, and specifically discloses a bioluminescent detection composition, a kit and a detection method for NAMPT enzyme activity.

Background technique

Nicotinamide mononucleotide adenylyltransferase (NAMPT) is the rate-limiting enzyme for the synthesis of nicotinamide adenine dinucleotide (NAD+). It is of great value in the fields of detection and evaluation, drug efficacy evaluation, and drug development. Nicotinamide (NAM) is catalyzed by NAMPT to synthesize nicotinamide mononucleotide (NMN), and NMN can be further converted by nicotinamide nucleotide adenosyltransferase (NMNAT) to produce NAD+. Studies have shown that NAMPT agonists have a good neuroprotective effect, and drug activation of NAMPT can increase the ability and concentration of cellular NAD+ synthesis, which is expected to become a strategy for the treatment of neurological injury-related diseases such as stroke. NAMPT expression, plasma concentration, enzyme activity, and NAD+ metabolism in a broader sense are potentially related to the survival ability of nerve cells, the recovery ability of cardiovascular and cerebrovascular diseases, and the cognitive ability of the body, so they have important research value. At the same time, clinical studies have found that in the early stages of some malignant cancers such as bladder cancer, colorectal cancer, breast cancer, prostate cancer, gastric cancer, etc., the expression of NAMPT will increase sharply, so the detection of NAMPT in serum can be used as a biomarker for bladder cancer. At the same time, it can also be used as an independent indicator for the prediction of non-muscle invasive bladder cancer.

NAMPT is ubiquitous in mammalian tissues, mainly in fat, liver, muscle and bone marrow. NAMPT is also distributed in peripheral blood leukocytes.

The current detection methods of NAMPT include colorimetric method, western blotting method, enzyme-linked immunosorbent assay method and so on. The above method is mainly used in the qualitative or semi-quantitative detection of NAMPT. The principle of the colorimetric method is to couple the two reactions catalyzed by NAMPT and NMNAT to finally generate NAD+, then further reduce NAD+ to NADH by alcohol dehydrogenase, and finally form formaldehyde in tetrazolium (WST-1) aqueous solution Tetrazolium. The formazolium tetrazolium molecule has a strong absorption light at 450 nm, and the measurement of the absorption light by a microplate reader can indirectly characterize the NAMPT activity of the analyte. Western blotting and ELISA both utilize antigen-antibody binding to measure NAMPT content through an enzyme cascade reaction. At present, the quantification of NAMPT in clinical samples mainly adopts enzyme-linked immunosorbent assay method.

There are two technical difficulties in the detection of NAMPT biomass in biological samples: (1) the sensitivity of existing NAMPT detection methods is limited; (2) the existing NAMPT enzyme activity detection is difficult to detect NAMPT activity in biological samples such as serum.

Based on the colorimetric NAMPT enzyme activity test, the enzymatic activity of NAMPT is evaluated by detecting the NAD+ content generated by the NAMPT rate-limiting coupled enzymatic reaction per unit time. Due to the limited sensitivity of the colorimetric method, the detection limit of this method is high, and the measurement of NAMPT activity at low concentrations cannot be completed. Since the detection uses a colorimetric method to quantify the concentration of enzymatic reaction products, it cannot directly measure the NAMPT enzyme activity in biological samples such as plasma and cell lysates, and needs to be combined with immunoprecipitation to separate the interfering matrix before the detection of cells The activity of NAMPT in the lysate makes the method complex and requires a long detection time. At the same time, because the interfering substance NADH may exist in biological samples, this method cannot completely exclude the influence of NADH on the detection accuracy. On the other hand, the method's reliance on immunoprecipitation pretreatment can lead to the loss of sample NAMPT during pretreatment, leading to distortion of NAMPT activity detection in biological samples.

The detection of NAMPT based on Western blot and enzyme-linked immunosorbent assay is more sensitive, and the detection line of NAMPT can be reduced to about 1 ng. However, this method can only detect the absolute content of NAMPT, but cannot detect the enzymatic activity of NAMPT. NAMPT with catalytic function requires a strict protein conformation, and aging, complex body fluid and cell fluid environment, or certain gene defects may lead to misfolding of NAMPT conformation, making simple NAMPT content detection useless and unable to reflect A more biologically meaningful indicator of NAMPT enzyme activity.

In addition, none of the methods in the prior art can realize real-time monitoring of NAMPT enzyme activity, and this limitation has a great impact on drug research related to NAD+ metabolism and NAMPT enzyme activity.

technical problem

None of the methods in the prior art can realize real-time monitoring of NAMPT enzyme activity, and this limitation has a great impact on drug research related to NAD ⁺ metabolism and NAMPT enzyme activity. The invention utilizes the NAD+ bioluminescence probe to measure the NAD+ concentration produced in the NAMPT and NMNAT enzyme coupling reaction system in real time. Since NAMPT is a rate-determining step in the reaction, the generation rate of NAD+ directly reflects the enzymatic activity of NAMPT. At the beginning of the reaction, NAMPT in the analyte converts NAM to NMN. NMN is then immediately converted to NAD+ by excess NMNAT. NAD+ concentrations are measured in real time by NAD+ bioluminescent probes.

technical solution

One aspect of the present invention provides a bioluminescence detection composition for NAMPT enzyme activity, the bioluminescence detection composition comprises NAM, PRPP, ATP, NMNAT, NAD+bioluminescence probe and corresponding bioluminescence probe substrate.

In the technical solution of the present invention, NAM, 5-phosphoribosyl-1-pyrophosphate (PRPP), adenosine triphosphate (ATP), NMNAT, NAD+bioluminescent probes and corresponding bioluminescent probe substrates are placed separately.

In the technical solution of the present invention, the NAD+ bioluminescence probe is a semi-synthetic NAD bioluminescence probe or a whole-gene encoded protein probe. As a preferred solution, the amino acid sequence of the whole-gene encoded protein probe is: SEQ ID NO .any of 1-5.

In the technical solution of the present invention, the corresponding bioluminescence probe substrate is selected from Furimazine.

In the technical solution of the present invention, the bioluminescent detection composition further includes a reaction solution, the reaction solution is preferably a buffer, more preferably 50 mM HEPES, 100 mM NaCl, 1 mM tris(2-chloroethyl) phosphate (TCEP), 5 mM MgCl2, pH 7.2 reaction solution.

In the technical solution of the present invention, the concentration of NAM in the bioluminescent detection composition is 10-200 μM, preferably 100 μM.

In the technical scheme of the present invention, the concentration of PRPP in the bioluminescent detection composition is 10-200 μM, preferably 100 μM.

In the technical scheme of the present invention, the concentration of ATP in the bioluminescent detection composition is 0.12-2 mM, preferably 2 mM.

In the technical solution of the present invention, the concentration of NMNAT in the bioluminescent detection composition is 0.1-10 mM, preferably 5 mM

In the technical solution of the present invention, the concentrations of NAD+ bioluminescence probe and the corresponding bioluminescence probe substrate in the bioluminescence detection composition are respectively 0.2-4 nM, preferably 4 nM.

Another aspect of the present invention provides a bioluminescence detection kit for NAMPT enzyme activity, the kit includes the above-mentioned bioluminescence detection composition.

Another aspect of the present invention provides a bioluminescence detection method for NAMPT enzyme activity, which comprises using the above-mentioned bioluminescence detection composition or bioluminescence detection kit to detect the luminescence intensity of the sample to be detected, and calculate the NAD+ concentration and use linear regression to determine the reaction The NAD+ production rate in the system, that is, the NAMPT enzyme activity.

In the technical solution of the present invention, the bioluminescence detection method comprises the following steps:

1) preparing a reaction solution, the reaction solution comprising the bioluminescence detection composition of the above-mentioned NAMPT enzyme activity;

2) Mix the test sample with the above reaction solution;

3) Measure the above mixture at 440 nm and 580 nm The dynamic change of the luminous intensity of nm two wavelengths;

4) Using 440 nm and 580 Calculate the NAD+ concentration at each moment in the reaction system by using the light intensity ratio of nm two wavelengths and the standard curve;

5) Plot NAD+ with the reaction time, and use linear regression to determine the NAD+ generation rate in the reaction system in M/min, and use this to characterize the NAMPT enzyme activity.

In the technical solution of the present invention, in step 3), the bioluminescent detection function of the microplate reader is used for detection.

In the technical solution of the present invention, the calculation equation in step 4) is as follows:

([NAD ⁺ ]=C ₅₀ ×(R _max -R/RR _min ) ^1/h

Where R is the light intensity ratio of 440 nm and 580 nm wavelengths measured by the microplate reader at each moment, R _max and R _min are the theoretical maximum and minimum light intensity ratios of the probe obtained through the standard curve, respectively, and C50 is the trigger 50 % is the analyte concentration constant for the probe signal change, and h is the Hill-coefficient of the fitted curve.

In the technical solution of the present invention, the test sample of the luminescence detection method is not pretreated; or no anticoagulant or NADH needs to be removed.

In the technical solution of the present invention, the test samples of the luminescence detection method are blood, plasma or serum, tissue homogenate samples, and enzyme activity test samples for drug screening.

Another aspect of the present invention provides a use of the above-mentioned bioluminescence detection composition or bioluminescence detection kit in the preparation of a reagent for evaluating the efficacy of a drug regulating NAMPT enzyme activity.

Another aspect of the present invention provides a use of the above-mentioned bioluminescence detection composition or bioluminescence detection kit in the preparation of reagents for high-throughput screening of NAMPT inhibitors or activators.

Specifically, the NAMPT activity detection reaction solution contains recombinant NMNAT, NAM, PRPP, ATP, NAD+bioluminescent probe and bioluminescent probe substrate. When measuring NAMPT activity, add the sample to be tested into the reaction solution, mix well, put it into a microplate reader, and use the bioluminescence detection function to measure 440 nm and 580 nm. The dynamic change of the luminous intensity of two nm wavelengths lasts for 15 min. After the measurement, use the light intensity ratio of 440 nm and 580 nm two wavelengths and the standard curve to calculate the NAD+ concentration in the reaction system at each moment. The NAD+ was plotted against the reaction time, and the NAD+ production rate in the reaction system was determined by linear regression, the unit was M/min, and the NAMPT enzyme activity was characterized by this.

Beneficial effect

1) The present invention realizes the direct, real-time and interference-free measurement of NAD ⁺ concentration in the NAMPT enzyme activity detection system. The present invention uses NAD ⁺ bioluminescence probe in the NAMPT-NMNAT coupling enzymatic reaction system, so that the NAD ⁺ generation rate limited by NAMPT can be monitored in real time by bioluminescence method. In the traditional method, NAD ⁺ produced by NAMPT and NMNAT coupling enzymatic reaction needs to go through two additional steps: i) alcohol dehydrogenase to reduce NAD ⁺ to NADH, ii) NADH to tetrazole (WST-1) Reduction to formazolium tetrazole can finally characterize the activity of NAMPT by detecting the absorbance of formazolium tetrazolium.

2) The NAD ⁺ in the system will not be consumed due to the existence of the probe, and will not affect the balance of the original enzymatic reaction, making the enzymatic reaction rate more real and reliable. However, the traditional detection method needs to convert all the reaction product NAD ⁺ into NADH, which will seriously change the balance of the enzymatic reaction.

3) The sensitivity of the NAD ⁺ bioluminescent probe is significantly higher than that of the absorbance colorimetric method, and the invention can realize the detection of NAMPT enzyme activity as low as 0.2 nM. Due to the more sensitive NAD ⁺ detection method, the present invention greatly reduces the time cost of NAMPT activity detection, from 30-60 min of traditional reaction to about 10 min.

4) The anti-interference ability of the bioluminescence probe is obviously better than that of the absorbance colorimetry. Since many matrix components in biological samples may absorb light, they have strong interference with traditional light absorption colorimetry. However, bioluminescent probes are based on the principle of luminescence, and the luminescence interference of biological samples is zero. It is not affected by other anticoagulants such as sodium citrate and heparin, nor by NADH in the blood.

5) Because the traditional absorbance colorimetric method is interfered by the sample matrix, the pretreatment of the sample is strictly required. Usually, it needs to be combined with the immunoprecipitation method to separate the interfering matrix before the quantification can be completed, which makes the overall operation cumbersome and time-consuming. However, NAMPT activity detection based on NAD ⁺ luminescent probe can avoid such pretreatment, and can directly quantitatively detect NAMPT enzyme activity in plasma and other biological samples. It can be used for drug efficacy evaluation of NAMPT enzyme activity regulating drugs and high-throughput screening of NAMPT inhibitors or activators.

Description of drawings

Figure 1: Colorimetric detection of NAMPT activity.

Figure 2: Detection of NAMPT by biological probe method.

Figure 3: Quantitative detection of NAMPT activity by NAD ⁺ bioluminescence probe.

Figure 4: Quantitative measurement of NAMPT activity in plasma.

Figure 5: Quantitative assessment of NAMPT-modulating drug effects.

Figure 6: Heat map of NAD ⁺ yield ratio produced by natural drug molecules regulating NAMPT activity.

Embodiments of the present invention

In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention will be described in detail below, but they should not be construed as limiting the scope of implementation of the present invention.

In a specific embodiment of the present invention, the NAD ⁺ bioluminescent probe can use any NAD ⁺ bioluminescent probe, for example, select the semi-synthetic NAD ⁺ bioluminescent probe that has been disclosed in the prior art or obtain it from the inventor's laboratory. The bioluminescent probe encoded by the whole gene is shown in the sequence SEQ ID NO.1-5 of the present invention.

Example 1. Real-time quantitative monitoring of low concentration NAMPT enzyme activity.

The present invention utilizes NAD ⁺ bioluminescence probe (bioluminescence resonance energy transfer probe, BRET) to rapidly and quantitatively detect the biological activity of NAMPT. The present invention characterizes the biological activity of NAMPT according to the production of NAD ⁺ by enzyme-linked reaction. The biological enzyme-linked reaction on which the present invention is based is shown in the technical roadmap 2.

The NAD ⁺ bioluminescent probe is a protein probe encoded by the whole gene, and its amino acid sequence is SEQ ID NO.1-5,

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLAD FKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATR GDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPGDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINEPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.1

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLAD FKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATR GDGTVGENITENLRTVRSVPMPLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPGDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINEPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.2

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLAD FKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATR GDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINEPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.3

ASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAA NYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDKLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGEN ITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHF KVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.4

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLAD FKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLLTLTTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATR GDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPPPATHELHIFGSINGVDFDM VGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHS KTELNFKEWQKAFTDVMGMDELYK SEQ ID NO.5

The corresponding substrate is Furimazine.

The reaction solution contains 50mM HEPES, 100 mM NaCl, 1 mM TCEP, 5 mM MgCl ₂ , pH 7.2; substrate: 100 μM NAM (nicotinamide, NAM), 100 μM PRPP (phosphoribosyl pyrophosphate), 2 mM ATP (adenosine triphosphate); NMNAT 5 mM, different concentrations of NAMPT (0.1 nmol/L, 0.2 nmol/L, 0.4 nmol/L, 0.6 nmol/L, 1 nmol/L, 2 nmol/L, 4 nmol/L, 6 nmol/L), 4 nM NAD ⁺ bioluminescent probe and its substrate. The reaction temperature was 37°C. The microplate reader continued to monitor the dynamic changes of luminous intensity at 440 nm and 580 nm for 20 minutes with a time interval of 1 minute. After the measurement, use the light intensity ratio of the two wavelengths of 440 nm and 580 nm and the standard curve to calculate the NAD ⁺ concentration at each moment in the reaction system. The calculation equation is as follows:

([NAD ⁺ ]=C ₅₀ ×(R _max -R/RR _min ) ^1/h

Where R is the light intensity ratio of 440 nm and 580 nm wavelengths measured by the microplate reader at each moment, R _max and R _min are the theoretical maximum and minimum light intensity ratios of the probe obtained through the standard curve, respectively, and C ₅₀ is the trigger The analyte concentration constant for 50% probe signal change, h is the Hill-coefficient of the fitted curve. The NAD ⁺ was plotted against the reaction time, and the NAD ⁺ production rate in the reaction system was determined by linear regression, in M/min, to characterize the NAMPT enzyme activity.

The experimental results are shown in Figure 3. The concentration of NAD ⁺ generated by the reaction system increases with time, which reflects that the detection system of the present invention can dynamically detect the reactivity of NAMPT in real time. Other probes have been tested to show similar effects. From the comparison of the curves of different concentrations of NAMPT, the higher the concentration of NAMPT, the larger the slope of the curve, indicating that the detection concentration range of the reaction system of the present invention is wider. At the same time, the reaction system can clearly detect the enzyme activity of NAMPT at concentrations above 0.2 nmol/L.

Example 2. Quantitative measurement of NAMPT enzymatic activity in unfractionated plasma samples.

Take 1 mL of fresh blood, centrifuge at 3000 rpm, 4 ℃ for 10 min. The supernatant is plasma. The collected plasma was quick-frozen in liquid nitrogen and stored in a -80°C refrigerator, or the enzyme activity of NAMPT in the plasma was directly detected. The reaction solution contained 50 mM HEPES, 50 mM NaCl, 1 mM TCEP, 5 mM MgCl ₂ , 100 μM NAM, 100 μM PRPP, 2 mM ATP, 5 mM NMNAT, 10 μL plasma sample, 4 nM NAD ⁺ bioluminescent probe and its Substrate, pH = 7.2; reaction temperature 37 °C. Use a microplate reader to continuously monitor the changes in luminous intensity at 440 nm and 580 nm with a time interval of 1 min. As shown in Figure 4, 0.6 nM NAMPT was used as the positive control group (the reaction system in Example 1); FK866 was a known and reported NAMPT inhibitor, and 1 μM FK866 was added to NAMPT as the negative control group.

The results are shown in Figure 4, and the results show that the NAMPT activity in the unseparated plasma sample can be quantitatively measured by the present invention. Although there are many interfering components in the plasma, the method of the present invention can provide results similar to parallel experiments in vitro.

Example 3. A detection method for quantitatively evaluating the effect of NAMPT-regulated drugs.

As an inhibitor of NAMPT, FK866 has good in vivo activity and is a potential drug molecule for the treatment of leukemia. SBI-797812, an activator of NAMPT, is one of the few potential drug molecules reported to directly enhance the activity of NAMPT. This example quantitatively evaluates the regulatory effect of such drugs on NAMPT activity. Experimental method: The reaction solution (pH 7.2) contains 50 mM HEPES, 50 mM NaCl, 1mM TCEP, 5 mM MgCl ₂ , 100 μM NAM, 100 μM PRPP, 2 mM ATP, 5 mM NMNAT, 1 nM NAMPT, 4 nM NAD ⁺ Bioluminescent probes and their substrates; different types and different concentrations of drug molecules were added to the reaction solution (see Figure 5 for specific information), the reaction temperature was 37 °C, and incubated for 1 h. Monitor the luminous intensity at 440 nm and 580 nm with a microplate reader. Calculate the 440 nm/580 nm light intensity ratio to calculate the NAD ⁺ production. In the present invention, the NAD ⁺ bioluminescence probe can be used to well evaluate the effects of FK866, P7C3-A20 and SBI-797812 on NAMPT activity. As shown in Figure 5, at 1 nM NAMPT at 37°C, the inhibitory effect of FK866 on NAMPT was verified by using NAD ⁺ bioluminescence probe; 1 μM SBI-797812 could increase the activity of NAMPT, and 10 μM SBI-797812 could inhibit NAMPT activity; P7C3-A20 had no significant effect on the activity of NAMPT. The invention can quantitatively evaluate the molecular ability of drugs regulating NAMPT activity.

Example 4. Screening method for NAMPT-regulated drugs based on enzyme activity.

Experimental method, the reaction solution (pH 7.2) contains 50 mM HEPES, 50 mM NaCl, 1 mM TCEP, 5 mM MgCl ₂ , 100 μM NAM, 100 μM PRPP, 2 mM ATP, 5 mM NMNAT, 1 nM NAMPT, 4 nM NAD ⁺ Bioluminescence probe; 1 μM drug molecules were added to the reaction solution, the reaction temperature was 37 °C, and incubated for 1 h. Monitor the light intensity at 440 nm and 580 nm with a microplate reader. Calculate the 440 nm/580 nm ratio and calculate the NAD ⁺ production. Finally, calculate the ratio (Ratio) of NAD ⁺ production in the experimental group compared to the control group (no drug molecules). When Ratio>1, the drug is a potential NAMPT activator; when Ratio<1, it is a potential NAMPT inhibitor; if Ratio=1, the drug does not significantly affect the NAMPT-catalyzed NAD ⁺ generation rate. The experimental results are shown in Figure 6, which shows a heat map of NAD ⁺ production (characterized by the NAD ⁺ yield ratio) by natural drug molecules regulating NAMPT activity. The invention can be used as a biotechnological means for high-throughput screening of NAMPT activators and inhibitors, using NAD ⁺ bioluminescent probes to screen drug molecules regulating NAMPT activity in natural drug molecule libraries. The method of the invention can reduce the time for drug molecules of NAMPT to combine with each other, and shorten the combined detection of NAMPT activators or inhibitors from a two-step method of thermal shift assay (Thermol shift assay) and colorimetry to a one-step method. Direct screening of activators or inhibitors of NAMPT by measuring NAD ⁺ production efficiency

Claims

A bioluminescence detection composition for NAMPT enzyme activity, characterized in that the bioluminescence detection composition comprises nicotinamide (NAM), 5-phosphoribosyl-1-pyrophosphate (PRPP), adenosine triphosphate (ATP), nicotinamide Nucleotide adenylyltransferase-1 (NMNAT), nicotinamide adenine dinucleotide (NAD + ) bioluminescent probes and corresponding bioluminescent probe substrates.
The bioluminescent detection composition according to claim 1, wherein the bioluminescent detection composition also comprises a reaction solution, the reaction solution is preferably a buffer, more preferably 50 mM HEPES, 100 mM NaCl, 1 mM Tris (2-Chloroethyl)phosphate (TCEP), 5 mM MgCl 2 , pH 7.2 reaction solution.
The bioluminescence detection composition according to claim 1, wherein the concentration of NAM in the bioluminescence detection composition is 10-200 μM;

Preferably, the concentration of PRPP in the bioluminescent detection composition is 10-200 μM;

Preferably, the concentration of NMNAT in the bioluminescent detection composition is 0.1-10 mM;

Preferably, the concentration of NMNAT in the bioluminescent detection composition is 0.1-10 mM;

Preferably, the concentrations of the NAD + bioluminescence probe and the corresponding bioluminescence probe substrate in the bioluminescence detection composition are 0.2-4 nM, respectively.
The bioluminescence detection composition according to claim 1, wherein the NAD + bioluminescence probe is a semi-synthetic NAD + bioluminescence probe or a whole gene encoded protein probe.
A bioluminescence detection kit for NAMPT enzyme activity, characterized in that the kit includes the above-mentioned bioluminescence detection composition.
A bioluminescent detection method for NAMPT enzyme activity, characterized in that it comprises the use of the bioluminescent detection composition described in any one of claims 1-4 or the bioluminescent detection kit described in claim 5 to detect the sample to be detected Luminescence intensity detection, and calculation of NAD + concentration, using linear regression to determine the NAD + generation rate in the reaction system, that is, NAMPT enzyme activity;
The bioluminescence detection method according to claim 6, characterized in that it comprises the following steps:

1) preparing a reaction solution, the reaction solution comprising the bioluminescence detection composition for NAMPT enzyme activity according to any one of claims 1-4;

2) Mix the test sample with the above reaction solution;

3) Measure the above mixture at 440 Dynamic change of luminous intensity at two wavelengths of nm and 580 nm;

4) Calculate the NAD + concentration at each moment in the reaction system by using the light intensity ratio of the two wavelengths of 440 nm and 580 nm and the standard curve;

5) Plot NAD + with reaction time, and use linear regression to determine the NAD + generation rate in the reaction system in M/min, and use this to characterize NAMPT enzyme activity;

Preferably, in step 3), the bioluminescence detection function of a microplate reader is used for detection.
The bioluminescence detection method according to claim 6 or 7, wherein the sample to be tested in the luminescence detection method is not pretreated; or no anticoagulant or NADH needs to be removed;

Preferably, the samples to be tested in the luminescent detection method are blood, plasma or serum, tissue homogenate samples, and enzyme activity test samples for drug screening.
The bioluminescent detection method according to claim 7, wherein the calculation equation in step 4) is as follows:

([NAD + ]=C 50 ×(R max -R/RR min ) 1/h

Where R is the light intensity ratio of 440 nm and 580 nm wavelengths measured by the microplate reader at each moment, R max and R min are the theoretical maximum and minimum light intensity ratios of the probe obtained through the standard curve, respectively, and C 50 is the trigger The analyte concentration constant for 50% probe signal change, h is the Hill-coefficient of the fitted curve.
The use of the bioluminescence detection composition according to any one of claims 1-4 or the bioluminescence detection kit according to claim 5 in the preparation of reagents for drug efficacy evaluation of NAMPT enzyme activity regulating drugs, or in the preparation of NAMPT inhibitory Use in reagents for high throughput screening of agents or activators.