Sialidase inhibitor time-resolved fluorescence screening method based on thermally activated delayed fluorescence probe
The organic small molecule thermally activated delayed fluorescence probe screening method solves the problems of high cost, high toxicity and complicated operation of lanthanide elements in the existing technology, and realizes rapid and accurate screening of sialidase inhibitors. It has wide applicability, low cost and non-toxicity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2026-03-31
AI Technical Summary
Current time-resolved fluorescence techniques for lanthanides are expensive, highly toxic, and cumbersome to operate, limiting their application in the screening of sialidase inhibitors.
A time-resolved fluorescence screening method for sialidase inhibitors based on organic small molecule thermally activated delayed fluorescence probes was adopted. The compound of formula (I) was used as a molecular probe. By specifically recognizing and blocking the intramolecular charge transfer process through sialidase, a significant fluorescence intensity and long fluorescence lifetime signal were generated. Combined with time-gating technology, interference from short-lifetime strong fluorescence signals was filtered out.
It achieves rapid, simple, non-toxic, and low-cost screening of sialidase inhibitors with high accuracy, wide applicability, avoids interference from short-lived strong fluorescence signals, and is easy to operate.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of enzyme inhibitor screening methods, specifically to the construction and application of a sialidase-responsive organic small molecule thermally activated delayed fluorescent probe and a time-resolved fluorescence screening method for sialidase inhibitors. Background Technology
[0002] Immune escape is a crucial means for pathogens to invade host cells and a significant pathway for the development and metastasis of malignant tumors. Therefore, exploring and inhibiting key biological factors in the immune escape process is of great importance for combating pathogenic infections and for anti-tumor therapy. It has been reported that high sialylation of the cell surface is one of the effective ways for pathogens and tumor cells to evade the immune response. Sialidase, as one of the key enzymes involved in the cell surface sialylation process, has recently been developed as a potential drug target for inhibiting immune escape. There is an urgent need to develop new sialylase inhibitors and screening tools for rapidly assessing sialylase activity in complex systems. Such screening tools will facilitate the rapid and accurate detection of sialylase activity and further identify effective inhibitors.
[0003] Fluorescence analysis technology has become a powerful tool for high-throughput screening of lead compounds and optimization of candidate drugs, offering advantages such as sensitivity, real-time detection, and non-invasiveness. Based on the different fluorescence parameters detected, fluorescence analysis techniques can be categorized into fluorescence intensity methods, fluorescence polarization methods, time-resolved fluorescence methods, fluorescence resonance energy transfer methods, and fluorescence lifetime methods. Among these, time-resolved fluorescence methods simultaneously detect both fluorescence wavelength and time parameters for signal resolution, offering advantages such as high throughput, avoidance of interference from short-lived strong fluorescence signals, and low false positive and false negative rates. For example, time-resolved fluorescence immunoassay utilizes lanthanide elements to label antigens or antibodies and measures fluorescence using time-resolved techniques based on the luminescence characteristics of lanthanide chelates, making it one of the most commonly used methods for small molecule screening. However, the high cost and toxicity of lanthanides, along with the cumbersome operation of immunoassay, limit the further application of this technology. Therefore, the development of rapid, simple, and metal-free time-resolved drug screening analysis techniques is urgently needed. Although challenging, this may inspire more effective and cost-efficient small molecule screening methods in the future. Summary of the Invention
[0004] To address the problems of high cost, high toxicity, and cumbersome operation of existing time-resolved fluorescence techniques using lanthanides, this invention aims to provide a time-resolved fluorescence screening method for sialidase inhibitors based on organic small molecule thermally activated delayed fluorescence probes. This method is simple to operate, non-toxic, rapid, low-cost, avoids interference from short-lived strong fluorescence signals, and has high accuracy.
[0005] The technical problem to be solved by this invention is achieved through the following technical solution:
[0006] A time-resolved fluorescence screening method for sialidase inhibitors based on thermally activated delayed fluorescent probes of organic small molecules is characterized by comprising a first reaction solution, a second reaction solution, and a diluent. The first reaction solution is a sialidase stock solution, wherein the solution is deionized water. The second reaction solution is an organic solution of the fluorescent probe shown in formula (I), wherein the organic solvent is dimethyl sulfoxide (DMSO). The diluent is a sodium acetate solution at pH 5.0.
[0007]
[0008] Furthermore, the first reaction solution, the second reaction solution, and the diluent are characterized in that the concentration of sialidase in the first reaction solution is 1-30 U / mL, preferably 3 U / mL; the concentration of the fluorescent probe shown in formula (I) in the second reaction solution is 1 mM; and the diluent contains 100 mM sodium acetate and 2 mM calcium chloride.
[0009] On the one hand, in the time-resolved fluorescence screening method for sialidase inhibitors based on thermally activated delayed fluorescent probes of organic small molecules described in this invention, the detection principle of the fluorescent probe is as follows:
[0010]
[0011] In the time-resolved fluorescence screening method for sialidase inhibitors described in this invention, the compound shown in formula (I) is used as a molecular probe. By linking the substrate sialic acid molecule, which can be specifically recognized by sialidase, to a fluorescent nucleus of formula (II) with thermally activated delayed fluorescence properties, the intramolecular charge transfer process of the fluorescent nucleus is blocked, thereby quenching the fluorescence intensity and fluorescence lifetime signals. After the fluorescent probe specifically reacts with sialidase, the fluorescence intensity is significantly enhanced and accompanied by a fluorescence lifetime on the order of microseconds. If a sialidase inhibitor is present in the system, the strength of the fluorescence intensity signal varies with the magnitude of the inhibition, but a long fluorescence lifetime signal is still present. Therefore, by setting a time gating, the fluorescence intensity signal after a specific time can be collected, thereby filtering out short-lived strong fluorescence signals that overlap with the emission peak of the fluorescent nucleus of formula (II), improving the applicability and accuracy of the method.
[0012] Furthermore, the lifetime of the solution containing the fluorophore of formula (II) generated after the molecular probe of formula (I) is reacted with sialidase is 19.3 μs.
[0013] On the other hand, the operation steps of the time-resolved fluorescence screening method for sialidase inhibitors based on organic small molecule thermally activated delayed fluorescent probes described in this invention are as follows:
[0014] (1) Measure the delayed fluorescence signal of the control solution.
[0015] The first reaction solution was diluted to 0.03 U / mL using a diluent. Then, 1.0–2.5 μL of the second reaction solution, preferably 1.0 μL, was added to the system. After incubation at 37°C for 10 min, the delayed fluorescence emission signal in the range of 400 nm to 800 nm was measured using an ELISA reader with 350 nm as the excitation wavelength.
[0016] (2) Determine the delayed fluorescence signal of the drug solution containing sialidase inhibitor to be screened.
[0017] Add the sialidase inhibitor to be screened drug to the dilution system containing the same concentration of the first reaction solution as in step (1), incubate at 37°C for 2 hours, then add the same volume of the second reaction solution as in step (1), incubate at 37°C for 10 minutes, and then use an ELISA reader to measure the fluorescence signal at the same emission wavelength and the same delay time as in step (1) at the same excitation wavelength as in step (1).
[0018] (3) Identification of sialidase inhibitors
[0019] Compare the intensity of the delayed fluorescence signal measured in steps (1) and (2). If the fluorescence intensity measured in step (2) is lower than that measured in step (1), it indicates that the added drug is a sialidase inhibitor. The extent of the inhibitory effect of the sialidase inhibitor can be determined based on the degree of fluorescence intensity reduction.
[0020] Compared with existing technologies, the time-resolved fluorescence screening method for sialidase inhibitors based on thermally activated delayed fluorescent probes of organic small molecules described in this invention has the following advantages:
[0021] (i) The probe molecule is an organic small molecule probe that does not contain lanthanide elements, is non-toxic and low in cost.
[0022] (ii) The second reaction solution itself has no fluorescence, but it can emit strong fluorescence after reacting with sialidase. When there is a sialidase inhibitor, the fluorescence intensity decreases with the degree of inhibition. The fluorescence signal changes significantly and is easy to detect.
[0023] (iii) The probe molecule can only be activated by sialidase and is not affected by other redox substances, metal ions, or other enzymes.
[0024] (iv) The fluorescence generation reaction is fast, and obvious differences can be seen within 10 minutes, which significantly accelerates the screening speed of sialidase inhibitors.
[0025] (v) It is easy to operate, has no technical barriers, and is widely applicable.
[0026] (vi) By detecting delayed fluorescence signals, interference from short-lived strong fluorescence signals can be avoided, making it more applicable and more accurate. Attached Figure Description
[0027] Figure 1 This indicates the emission spectrum of the probe molecule before and after reacting with sialidase.
[0028] Figure 2 This represents the photoluminescence decay curve of the solution after the probe molecule reacts with sialidase.
[0029] Figure 3 This indicates that the probe molecule specifically recognizes sialidase.
[0030] Figure 4 This indicates the delayed emission spectra of the sialidase inhibitor candidate drug and probe molecules under 350 nm excitation.
[0031] Figure 5 The fluorescence emission spectra of the compound are shown at different delay times.
[0032] Figure 6 This indicates the results of the time-resolved fluorescence inhibitor screening.
[0033] Figure 7 The accompanying diagram represents a summary of the instruction manual. Detailed Implementation
[0034] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. Those skilled in the art should understand that any simple modifications or equivalent substitutions made without departing from the concept of the present invention should be considered to fall within the protection scope of the present invention.
[0035] Unless otherwise specified, all reagents described in this invention, such as sialidase, are commercially available. In the method, the fluorescence emission spectrum is measured using a Spark 10M multifunctional microplate reader platform (Tecan, Switzerland), and the photoluminescence decay curve is detected using an FLS980 Edinburgh fluorescence spectrometer (Edinburgh, UK).
[0036] Example 1: Detection of fluorescence signals before and after the reaction of probe molecules with sialidase
[0037] In the control group, 2.5 μL of DMSO and 5.0 μL of the second reaction solution were added to 492.5 μL of diluent. In the experimental group, the first reaction solution was diluted to 492.5 μL with diluent, and the sialidase concentration was 0.03 U / mL. Then, 2.5 μL of DMSO and 5.0 μL of the second reaction solution were added to the diluted solution. After incubation at 37 °C for 10 min, the samples were added to 96-well plates, 150 μL per well. The delayed fluorescence emission spectra of the control and experimental group samples in the range of 400 nm to 800 nm were measured under 350 nm excitation.
[0038] Experimental results are as follows Figure 1 As shown, the fluorescence of the second reaction solution, which is the control group, is very weak. Figure 1 (Solid line), and after reacting with sialidase, it can emit strong fluorescence ( Figure 1 (Dashed line), the fluorescence intensity changes significantly.
[0039] Example 2: Detection of photoluminescence attenuation signal in solution after the reaction of probe molecules with sialidase
[0040] The first reaction solution was diluted to 2955 μL with diluent, and the sialidase concentration was 0.03 U / mL. 15 μL of DMSO and 30 μL of the second reaction solution were added to bring the final probe molecule concentration to 10 μM. After incubation at 37°C for 30 min, the solution was added to a quartz dish, and the photoluminescence decay signal was measured using an FLS980 Edinburgh fluorescence spectrometer. The excitation wavelength was 377 nm, the emission wavelength was 540 nm, the laser model was VPL-375, the maximum average power was 100 mW, and the pulse width was 500 ns.
[0041] Experimental results are as follows Figure 2 As shown, the probe molecule produces a significant long fluorescence lifetime signal after reacting with sialidase. Figure 2 (Solid line).
[0042] Example 3: Probe molecules specifically recognize sialidase
[0043] In the control group, the first reaction solution was diluted to 492.5 μL with diluent to achieve a sialidase concentration of 0.03 U / mL. Then, 2.5 μL of DMSO and 5.0 μL of the second reaction solution were added. After incubation at 37°C for 10 min, 150 μL of the sample was added to each well of a 96-well plate. In the experimental group, different influencing factors were diluted to 492.5 μL with diluent at the following concentrations: 1 μg / mL nitroreductase, 0.03 U / mL β-galactosidase, 1 mM cysteine, 1 mM dithiothreitol, 1 mM glutathione, 1 mM arginine, 1 mM vitamin C, 10 mM glucose, 10 mM sodium hypochlorite, 10 mM hydrogen peroxide, 2.5 mM calcium chloride, 2.5 mM magnesium chloride, 10 mM potassium chloride, and 10 mM sodium chloride. Then, 2.5 μL of DMSO and 5.0 μL of the second reaction solution were added. After incubation at 37℃ for 10 min, the sample was added to a 96-well plate, 150 μL per well. No delayed fluorescence emission signal was observed in the control and experimental groups at 540 nm under 350 nm excitation.
[0044] like Figure 3As shown, the probe molecule can only be activated by sialidase to produce a strong fluorescent signal. Other influencing factors, including redox substances, metal ions, and other enzymes, cannot activate the probe molecule to produce a strong fluorescent signal.
[0045] Example 4: Detection of delayed fluorescence signal during sialidase inhibitor screening
[0046] In a 96-well plate, the control group diluted the first reaction solution to 98.5 μL with diluent to achieve a sialidase concentration of 0.03 U / mL. Then, 0.5 μL of DMSO and 1.0 μL of the second reaction solution were added. After incubation at 37 °C for 10 min, the delayed fluorescence emission spectra of the control group samples in the range of 400 nm to 800 nm were measured under 350 nm excitation.
[0047] In a 96-well plate, the experimental group diluted the first reaction solution to 98.5 μL with diluent, the sialidase concentration was 0.03 U / mL, and 0.5 μL of the sialidase inhibitor to be screened compound (DMSO as solvent) was added. After incubation at 37℃ for 2 h, 1.0 μL of the second reaction solution was added, and after incubation at 37℃ for 10 min, the delayed fluorescence emission spectrum of the experimental group sample in the range of 400 nm to 800 nm was measured under 350 nm excitation.
[0048] Experimental results are as follows Figure 4 As shown, the experimental group ( Figure 4 The fluorescence intensity of the (dashed line) group was much stronger than that of the control group. Figure 4 The solid line indicates that in the sialidase inhibitor screening experiment, the sialidase inhibitor to be screened drug showed a strong fluorescence signal that overlapped with the probe molecule, which seriously interfered with the detection of the probe molecule's fluorescence signal.
[0049] Experiment 5: Fluorescence emission signals of compounds at different delay times
[0050] In a 96-well plate, the control group was diluted with diluent to 98.5 μL of the first reaction solution to achieve a sialidase concentration of 0.03 U / mL. Then, 0.5 μL of DMSO and 1.0 μL of the second reaction solution were added. After incubation at 37 °C for 10 min, the fluorescence emission spectra of the control group samples were measured in the range of 400 nm to 800 nm under different time-gated conditions at 350 nm excitation.
[0051] In a 96-well plate, the experimental group diluted the first reaction solution to 98.5 μL with diluent to achieve a sialidase concentration of 0.03 U / mL. 0.5 μL of the screening drug Pomalidomide solution (DMSO as solvent) was added, and the plate was incubated at 37°C for 2 h. Then, 1.0 μL of the second reaction solution was added, and the plate was incubated at 37°C for another 10 min. Finally, the fluorescence emission spectra of the experimental group samples in the range of 400 nm to 800 nm under different time-gated conditions were measured under 350 nm excitation.
[0052] Experimental results are as follows Figure 5 As shown, with the increase of the delay time, the experimental group ( Figure 5 The fluorescence emission peak of the dashed line gradually converges with that of the control group ( Figure 5 The consistent fluorescence emission peak shape of the solid line indicates that the fluorescence emission peak of Pomalidomide itself gradually disappears with the increase of delay time in the experimental group, thus revealing the fluorescence emission peak of the covered probe molecule.
[0053] in accordance with Figure 5 The results show that the optimal gate time for fluorescence signal collection is 75 μs, at which point the influence of short-lived, strong-fluorescence sialidase inhibitors on the detection of probe molecule fluorescence signals can be completely eliminated.
[0054] Example 6: Screening for Time-Resolved Fluorescent Sialidase Inhibitors
[0055] In a 96-well plate, the control group was diluted with diluent to 98.5 μL of the first reaction solution to achieve a sialidase concentration of 0.03 U / mL. Then, 0.5 μL of DMSO and 1.0 μL of the second reaction solution were added. After incubation at 37 °C for 10 min, the fluorescence emission spectra of the control group samples were measured in the range of 400 nm to 800 nm at a 75 μs delay under 350 nm excitation.
[0056] In a 96-well plate, the experimental group diluted the first reaction solution to 98.5 μL with diluent to make the sialidase concentration 0.03 U / mL. 0.5 μL of the sialidase inhibitor compound solution to be screened (DMSO as solvent) was added. After incubation at 37°C for 2 h, 1.0 μL of the second reaction solution was added, and the plate was incubated at 37°C for another 10 min. Finally, the fluorescence emission spectrum of the experimental group samples in the range of 400 nm to 800 nm was measured at a 75 μs delay under 350 nm excitation.
[0057] Experimental results are as follows Figure 6As shown, the 75 μs delayed fluorescence emission spectrum avoids the influence of the short-lived, strongly fluorescent sialidase inhibitor compounds to be screened on the detection of the probe molecule's fluorescence signal. Among the five sialidase inhibitors to be screened—Pomalidomide, Daclatasvirdihydrochloride, Elbasvir, Rucaparib phosphate, and Pralatrexate—Pomalidomide ( Figure 6 (a dashed line) and Rucaparib phosphate ( Figure 6 The group represented by the dashed line (d) shows the difference between the control group and the control group ( Figure 6 a and Figure 6 The fluorescence signals (d solid line) are similar in intensity, therefore Pomalidomide and Rucaparibphosphate have no inhibitory effect on sialidase. Daclatasvir dihydrochloride ( Figure 6 The fluorescence signal of the probe molecules in group b (dashed line) was slightly lower than that in the control group. Figure 6 The fluorescent signal of the probe molecule (b solid line) indicates that Daclatasvir dihydrochloride has a relatively weak inhibitory effect on sialidase. Elbasvir ( Figure 6 c (dashed line) and Pralatrexate ( Figure 6 The probe molecules in the group represented by the dashed line (e) showed a difference compared to the control group ( Figure 6 c and Figure 6 The weaker fluorescence signal (e solid line) indicates that Elbasvir and Pralatrexate have a relatively stronger inhibitory effect on sialidase.
[0058] The above results, especially Figure 4 and Figure 6 This demonstrates that the time-resolved fluorescence screening method based on thermally activated delayed fluorescent probes of organic small molecules established in this invention can be successfully used for sialidase inhibitor screening.
Claims
1. A time-resolved fluorescence screening method for sialidase inhibitors based on organic small-molecule thermally activated delayed fluorescence probes, characterized in that, The first reaction solution is a sialidase stock solution, the solution is deionized water; the second reaction solution is an organic solution of the fluorescent probe shown in formula (I), the organic solvent is dimethyl sulfoxide (DMSO); the dilution solution is a pH 5.0 sodium acetate solution, 2. The screening method of claim 1, characterized in that, The concentration of sialidase in the first reaction solution is 1-30 U / mL, preferably 3 U / mL; the concentration of the fluorescent probe shown in formula (I) in the second reaction solution is 1 mM; the dilution solution contains 100 mM sodium acetate and 2 mM calcium chloride.
3. The screening method of any one of claims 1-2, comprising the following steps: (1) measuring the delayed fluorescence signal of the control solution The first reaction solution is diluted to a specific concentration using the dilution solution, then a certain volume of the second reaction solution is added to the system, and after incubation, the delayed fluorescence emission signal in the range of 400-800 nm is measured using an enzyme marker with 350 nm as the excitation wavelength; (2) measuring the delayed fluorescence signal of the sialidase inhibitor drug solution to be screened The sialidase inhibitor drug to be screened is added to the dilution system containing the first reaction solution with the same concentration as step (1), and the first stage incubation is carried out, then the second reaction solution with the same volume as step (1) is added, and the second stage incubation is carried out, and the delayed fluorescence signal at the same emission wavelength as step (1) is measured using an enzyme marker with the same excitation wavelength as step (1); (3) sialidase inhibitor determination Compare the intensity of the delayed fluorescence signals measured in steps (1) and (2), if the fluorescence intensity measured in step (2) is lower than that measured in step (1), it indicates that the drug is a sialidase inhibitor, and the degree of inhibition of the inhibitor on sialidase can be determined according to the degree of reduction of fluorescence intensity.
4. The screening method of claim 3, characterized in that, The specific concentration of the first reaction solution in the dilution system is 0.01-0.3 U / mL, preferably 0.03 U / mL.
5. The screening method of claim 3, wherein, The certain volume of the second reaction solution in the dilution system is 1.0-2.5 μL, preferably 1.0 μL.
6. The screening method of claim 3, wherein, The incubation conditions in step (1) are 37°C constant temperature for 10 min, and the first stage incubation conditions in step (2) are 37°C constant temperature for 2 h, and the second stage incubation conditions are 37°C constant temperature for 10 min.
7. The screening method of claim 3, wherein, The delayed fluorescence intensity is measured, characterized in that the delay time is 75 μs.